Publications
Preprints available on bioRxiv.
1.
Lisanza, Sidney Lyayuga; Gershon, Jacob Merle; Tipps, Samuel W. K.; Sims, Jeremiah Nelson; Arnoldt, Lucas; Hendel, Samuel J.; Simma, Miriam K.; Liu, Ge; Yase, Muna; Wu, Hongwei; Tharp, Claire D.; Li, Xinting; Kang, Alex; Brackenbrough, Evans; Bera, Asim K.; Gerben, Stacey; Wittmann, Bruce J.; McShan, Andrew C.; Baker, David
Multistate and functional protein design using RoseTTAFold sequence space diffusion Journal Article
In: Nature Biotechnology, 2024.
@article{Lisanza2024,
title = {Multistate and functional protein design using RoseTTAFold sequence space diffusion},
author = {Sidney Lyayuga Lisanza and Jacob Merle Gershon and Samuel W. K. Tipps and Jeremiah Nelson Sims and Lucas Arnoldt and Samuel J. Hendel and Miriam K. Simma and Ge Liu and Muna Yase and Hongwei Wu and Claire D. Tharp and Xinting Li and Alex Kang and Evans Brackenbrough and Asim K. Bera and Stacey Gerben and Bruce J. Wittmann and Andrew C. McShan and David Baker},
url = {https://www.nature.com/articles/s41587-024-02395-w, Nature Biotechnology [Open Access]},
doi = {10.1038/s41587-024-02395-w},
year = {2024},
date = {2024-09-25},
urldate = {2024-09-25},
journal = {Nature Biotechnology},
publisher = {Springer Science and Business Media LLC},
abstract = {Protein denoising diffusion probabilistic models are used for the de novo generation of protein backbones but are limited in their ability to guide generation of proteins with sequence-specific attributes and functional properties. To overcome this limitation, we developed ProteinGenerator (PG), a sequence space diffusion model based on RoseTTAFold that simultaneously generates protein sequences and structures. Beginning from a noised sequence representation, PG generates sequence and structure pairs by iterative denoising, guided by desired sequence and structural protein attributes. We designed thermostable proteins with varying amino acid compositions and internal sequence repeats and cage bioactive peptides, such as melittin. By averaging sequence logits between diffusion trajectories with distinct structural constraints, we designed multistate parent–child protein triples in which the same sequence folds to different supersecondary structures when intact in the parent versus split into two child domains. PG design trajectories can be guided by experimental sequence–activity data, providing a general approach for integrated computational and experimental optimization of protein function.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Protein denoising diffusion probabilistic models are used for the de novo generation of protein backbones but are limited in their ability to guide generation of proteins with sequence-specific attributes and functional properties. To overcome this limitation, we developed ProteinGenerator (PG), a sequence space diffusion model based on RoseTTAFold that simultaneously generates protein sequences and structures. Beginning from a noised sequence representation, PG generates sequence and structure pairs by iterative denoising, guided by desired sequence and structural protein attributes. We designed thermostable proteins with varying amino acid compositions and internal sequence repeats and cage bioactive peptides, such as melittin. By averaging sequence logits between diffusion trajectories with distinct structural constraints, we designed multistate parent–child protein triples in which the same sequence folds to different supersecondary structures when intact in the parent versus split into two child domains. PG design trajectories can be guided by experimental sequence–activity data, providing a general approach for integrated computational and experimental optimization of protein function.
2.
Moyer, Adam P.; Ramelot, Theresa A.; Curti, Mariano; Eastman, Margaret A.; Kang, Alex; Bera, Asim K.; Tejero, Roberto; Salveson, Patrick J.; Curutchet, Carles; Romero, Elisabet; Montelione, Gaetano T.; Baker, David
Enumerative Discovery of Noncanonical Polypeptide Secondary Structures Journal Article
In: Journal of the American Chemical Society, 2024.
@article{Moyer2024,
title = {Enumerative Discovery of Noncanonical Polypeptide Secondary Structures},
author = {Adam P. Moyer and Theresa A. Ramelot and Mariano Curti and Margaret A. Eastman and Alex Kang and Asim K. Bera and Roberto Tejero and Patrick J. Salveson and Carles Curutchet and Elisabet Romero and Gaetano T. Montelione and David Baker},
url = {https://pubs.acs.org/doi/full/10.1021/jacs.4c04991, LACS [Open Access]},
doi = {10.1021/jacs.4c04991},
year = {2024},
date = {2024-09-18},
urldate = {2024-09-18},
journal = {Journal of the American Chemical Society},
publisher = {American Chemical Society (ACS)},
abstract = {Energetically favorable local interactions can overcome the entropic cost of chain ordering and cause otherwise flexible polymers to adopt regularly repeating backbone conformations. A prominent example is the α helix present in many protein structures, which is stabilized by i, i + 4 hydrogen bonds between backbone peptide units. With the increased chemical diversity offered by unnatural amino acids and backbones, it has been possible to identify regularly repeating structures not present in proteins, but to date, there has been no systematic approach for identifying new polymers likely to have such structures despite their considerable potential for molecular engineering. Here we describe a systematic approach to search through dipeptide combinations of 130 chemically diverse amino acids to identify those predicted to populate unique low-energy states. We characterize ten newly identified dipeptide repeating structures using circular dichroism spectroscopy and comparison with calculated spectra. NMR and X-ray crystallographic structures of two of these dipeptide-repeat polymers are similar to the computational models. Our approach is readily generalizable to identify low-energy repeating structures for a wide variety of polymers, and our ordered dipeptide repeats provide new building blocks for molecular engineering.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Energetically favorable local interactions can overcome the entropic cost of chain ordering and cause otherwise flexible polymers to adopt regularly repeating backbone conformations. A prominent example is the α helix present in many protein structures, which is stabilized by i, i + 4 hydrogen bonds between backbone peptide units. With the increased chemical diversity offered by unnatural amino acids and backbones, it has been possible to identify regularly repeating structures not present in proteins, but to date, there has been no systematic approach for identifying new polymers likely to have such structures despite their considerable potential for molecular engineering. Here we describe a systematic approach to search through dipeptide combinations of 130 chemically diverse amino acids to identify those predicted to populate unique low-energy states. We characterize ten newly identified dipeptide repeating structures using circular dichroism spectroscopy and comparison with calculated spectra. NMR and X-ray crystallographic structures of two of these dipeptide-repeat polymers are similar to the computational models. Our approach is readily generalizable to identify low-energy repeating structures for a wide variety of polymers, and our ordered dipeptide repeats provide new building blocks for molecular engineering.
3.
Pillai, Arvind; Idris, Abbas; Philomin, Annika; Weidle, Connor; Skotheim, Rebecca; Leung, Philip J. Y.; Broerman, Adam; Demakis, Cullen; Borst, Andrew J.; Praetorius, Florian; Baker, David
De novo design of allosterically switchable protein assemblies Journal Article
In: Nature, 2024.
@article{Pillai2024,
title = {De novo design of allosterically switchable protein assemblies},
author = {Arvind Pillai and Abbas Idris and Annika Philomin and Connor Weidle and Rebecca Skotheim and Philip J. Y. Leung and Adam Broerman and Cullen Demakis and Andrew J. Borst and Florian Praetorius and David Baker},
url = {https://www.nature.com/articles/s41586-024-07813-2, Nature [Open Access]},
doi = {10.1038/s41586-024-07813-2},
year = {2024},
date = {2024-08-14},
urldate = {2024-08-14},
journal = {Nature},
publisher = {Springer Science and Business Media LLC},
abstract = {Allosteric modulation of protein function, wherein the binding of an effector to a protein triggers conformational changes at distant functional sites, plays a central part in the control of metabolism and cell signalling. There has been considerable interest in designing allosteric systems, both to gain insight into the mechanisms underlying such ‘action at a distance’ modulation and to create synthetic proteins whose functions can be regulated by effectors. However, emulating the subtle conformational changes distributed across many residues, characteristic of natural allosteric proteins, is a significant challenge. Here, inspired by the classic Monod–Wyman–Changeux model of cooperativity, we investigate the de novo design of allostery through rigid-body coupling of peptide-switchable hinge modules to protein interfaces that direct the formation of alternative oligomeric states. We find that this approach can be used to generate a wide variety of allosterically switchable systems, including cyclic rings that incorporate or eject subunits in response to peptide binding and dihedral cages that undergo effector-induced disassembly. Size-exclusion chromatography, mass photometry and electron microscopy reveal that these designed allosteric protein assemblies closely resemble the design models in both the presence and absence of peptide effectors and can have ligand-binding cooperativity comparable to classic natural systems such as haemoglobin. Our results indicate that allostery can arise from global coupling of the energetics of protein substructures without optimized side-chain–side-chain allosteric communication pathways and provide a roadmap for generating allosterically triggerable delivery systems, protein nanomachines and cellular feedback control circuitry.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Allosteric modulation of protein function, wherein the binding of an effector to a protein triggers conformational changes at distant functional sites, plays a central part in the control of metabolism and cell signalling. There has been considerable interest in designing allosteric systems, both to gain insight into the mechanisms underlying such ‘action at a distance’ modulation and to create synthetic proteins whose functions can be regulated by effectors. However, emulating the subtle conformational changes distributed across many residues, characteristic of natural allosteric proteins, is a significant challenge. Here, inspired by the classic Monod–Wyman–Changeux model of cooperativity, we investigate the de novo design of allostery through rigid-body coupling of peptide-switchable hinge modules to protein interfaces that direct the formation of alternative oligomeric states. We find that this approach can be used to generate a wide variety of allosterically switchable systems, including cyclic rings that incorporate or eject subunits in response to peptide binding and dihedral cages that undergo effector-induced disassembly. Size-exclusion chromatography, mass photometry and electron microscopy reveal that these designed allosteric protein assemblies closely resemble the design models in both the presence and absence of peptide effectors and can have ligand-binding cooperativity comparable to classic natural systems such as haemoglobin. Our results indicate that allostery can arise from global coupling of the energetics of protein substructures without optimized side-chain–side-chain allosteric communication pathways and provide a roadmap for generating allosterically triggerable delivery systems, protein nanomachines and cellular feedback control circuitry.
4.
Sun, Ke; Li, Sicong; Zheng, Bowen; Zhu, Yanlei; Wang, Tongyue; Liang, Mingfu; Yao, Yue; Zhang, Kairan; Zhang, Jizhong; Li, Hongyong; Han, Dongyang; Zheng, Jishen; Coventry, Brian; Cao, Longxing; Baker, David; Liu, Lei; Lu, Peilong
Accurate de novo design of heterochiral protein–protein interactions Journal Article
In: Cell Research, 2024.
@article{Sun2024,
title = {Accurate de novo design of heterochiral protein–protein interactions},
author = {Ke Sun and Sicong Li and Bowen Zheng and Yanlei Zhu and Tongyue Wang and Mingfu Liang and Yue Yao and Kairan Zhang and Jizhong Zhang and Hongyong Li and Dongyang Han and Jishen Zheng and Brian Coventry and Longxing Cao and David Baker and Lei Liu and Peilong Lu},
url = {https://www.nature.com/articles/s41422-024-01014-2, Cell Research [Open Access]},
doi = {10.1038/s41422-024-01014-2},
year = {2024},
date = {2024-08-14},
urldate = {2024-08-14},
journal = {Cell Research},
publisher = {Springer Science and Business Media LLC},
abstract = {Abiotic d-proteins that selectively bind to natural l-proteins have gained significant biotechnological interest. However, the underlying structural principles governing such heterochiral protein–protein interactions remain largely unknown. In this study, we present the de novo design of d-proteins consisting of 50–65 residues, aiming to target specific surface regions of l-proteins or l-peptides. Our designer d-protein binders exhibit nanomolar affinity toward an artificial l-peptide, as well as two naturally occurring proteins of therapeutic significance: the D5 domain of human tropomyosin receptor kinase A (TrkA) and human interleukin-6 (IL-6). Notably, these d-protein binders demonstrate high enantiomeric specificity and target specificity. In cell-based experiments, designer d-protein binders effectively inhibited the downstream signaling of TrkA and IL-6 with high potency. Moreover, these binders exhibited remarkable thermal stability and resistance to protease degradation. Crystal structure of the designed heterochiral d-protein–l-peptide complex, obtained at a resolution of 2.0 Å, closely resembled the design model, indicating that the computational method employed is highly accurate. Furthermore, the crystal structure provides valuable information regarding the interactions between helical l-peptides and d-proteins, particularly elucidating a novel mode of heterochiral helix–helix interactions. Leveraging the design of d-proteins specifically targeting l-peptides or l-proteins opens up avenues for systematic exploration of the mirror-image protein universe, paving the way for a diverse range of applications.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Abiotic d-proteins that selectively bind to natural l-proteins have gained significant biotechnological interest. However, the underlying structural principles governing such heterochiral protein–protein interactions remain largely unknown. In this study, we present the de novo design of d-proteins consisting of 50–65 residues, aiming to target specific surface regions of l-proteins or l-peptides. Our designer d-protein binders exhibit nanomolar affinity toward an artificial l-peptide, as well as two naturally occurring proteins of therapeutic significance: the D5 domain of human tropomyosin receptor kinase A (TrkA) and human interleukin-6 (IL-6). Notably, these d-protein binders demonstrate high enantiomeric specificity and target specificity. In cell-based experiments, designer d-protein binders effectively inhibited the downstream signaling of TrkA and IL-6 with high potency. Moreover, these binders exhibited remarkable thermal stability and resistance to protease degradation. Crystal structure of the designed heterochiral d-protein–l-peptide complex, obtained at a resolution of 2.0 Å, closely resembled the design model, indicating that the computational method employed is highly accurate. Furthermore, the crystal structure provides valuable information regarding the interactions between helical l-peptides and d-proteins, particularly elucidating a novel mode of heterochiral helix–helix interactions. Leveraging the design of d-proteins specifically targeting l-peptides or l-proteins opens up avenues for systematic exploration of the mirror-image protein universe, paving the way for a diverse range of applications.
5.
Jiang, Hanlun; Jude, Kevin M.; Wu, Kejia; Fallas, Jorge; Ueda, George; Brunette, T. J.; Hicks, Derrick R.; Pyles, Harley; Yang, Aerin; Carter, Lauren; Lamb, Mila; Li, Xinting; Levine, Paul M.; Stewart, Lance; Garcia, K. Christopher; Baker, David
De novo design of buttressed loops for sculpting protein functions Journal Article
In: Nature Chemical Biology, 2024.
@article{Jiang2024,
title = {De novo design of buttressed loops for sculpting protein functions},
author = {Jiang, Hanlun
and Jude, Kevin M.
and Wu, Kejia
and Fallas, Jorge
and Ueda, George
and Brunette, T. J.
and Hicks, Derrick R.
and Pyles, Harley
and Yang, Aerin
and Carter, Lauren
and Lamb, Mila
and Li, Xinting
and Levine, Paul M.
and Stewart, Lance
and Garcia, K. Christopher
and Baker, David},
url = {https://www.nature.com/articles/s41589-024-01632-2, Nature Chemical Biology [Open Access]
https://www.bakerlab.org/wp-content/uploads/2024/05/s41589-024-01632-2.pdf, PDF},
doi = {10.1038/s41589-024-01632-2},
year = {2024},
date = {2024-05-30},
urldate = {2024-05-30},
journal = {Nature Chemical Biology},
abstract = {In natural proteins, structured loops have central roles in molecular recognition, signal transduction and enzyme catalysis. However, because of the intrinsic flexibility and irregularity of loop regions, organizing multiple structured loops at protein functional sites has been very difficult to achieve by de novo protein design. Here we describe a solution to this problem that designs tandem repeat proteins with structured loops (9–14 residues) buttressed by extensive hydrogen bonding interactions. Experimental characterization shows that the designs are monodisperse, highly soluble, folded and thermally stable. Crystal structures are in close agreement with the design models, with the loops structured and buttressed as designed. We demonstrate the functionality afforded by loop buttressing by designing and characterizing binders for extended peptides in which the loops form one side of an extended binding pocket. The ability to design multiple structured loops should contribute generally to efforts to design new protein functions.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In natural proteins, structured loops have central roles in molecular recognition, signal transduction and enzyme catalysis. However, because of the intrinsic flexibility and irregularity of loop regions, organizing multiple structured loops at protein functional sites has been very difficult to achieve by de novo protein design. Here we describe a solution to this problem that designs tandem repeat proteins with structured loops (9–14 residues) buttressed by extensive hydrogen bonding interactions. Experimental characterization shows that the designs are monodisperse, highly soluble, folded and thermally stable. Crystal structures are in close agreement with the design models, with the loops structured and buttressed as designed. We demonstrate the functionality afforded by loop buttressing by designing and characterizing binders for extended peptides in which the loops form one side of an extended binding pocket. The ability to design multiple structured loops should contribute generally to efforts to design new protein functions.
6.
Craven, Timothy W.; Nolan, Mark D.; Bailey, Jonathan; Olatunji, Samir; Bann, Samantha J.; Bowen, Katherine; Ostrovitsa, Nikita; Costa, Thaina M. Da; Ballantine, Ross D.; Weichert, Dietmar; Levine, Paul M.; Stewart, Lance J.; Bhardwaj, Gaurav; Geoghegan, Joan A.; Cochrane, Stephen A.; Scanlan, Eoin M.; Caffrey, Martin; Baker, David
Computational Design of Cyclic Peptide Inhibitors of a Bacterial Membrane Lipoprotein Peptidase Journal Article
In: ACS Chemical Biology, 2024.
@article{Craven2024,
title = {Computational Design of Cyclic Peptide Inhibitors of a Bacterial Membrane Lipoprotein Peptidase},
author = {Timothy W. Craven and Mark D. Nolan and Jonathan Bailey and Samir Olatunji and Samantha J. Bann and Katherine Bowen and Nikita Ostrovitsa and Thaina M. Da Costa and Ross D. Ballantine and Dietmar Weichert and Paul M. Levine and Lance J. Stewart and Gaurav Bhardwaj and Joan A. Geoghegan and Stephen A. Cochrane and Eoin M. Scanlan and Martin Caffrey and David Baker},
url = {https://pubs.acs.org/doi/10.1021/acschembio.4c00076, ACS Chem. Bio. [Open Access]
https://www.bakerlab.org/wp-content/uploads/2024/05/craven-et-al-2024-computational-design-of-cyclic-peptide-inhibitors-of-a-bacterial-membrane-lipoprotein-peptidase.pdf, PDF},
doi = {10.1021/acschembio.4c00076},
year = {2024},
date = {2024-05-07},
urldate = {2024-05-07},
journal = {ACS Chemical Biology},
publisher = {American Chemical Society (ACS)},
abstract = {There remains a critical need for new antibiotics against multi-drug-resistant Gram-negative bacteria, a major global threat that continues to impact mortality rates. Lipoprotein signal peptidase II is an essential enzyme in the lipoprotein biosynthetic pathway of Gram-negative bacteria, making it an attractive target for antibacterial drug discovery. Although natural inhibitors of LspA have been identified, such as the cyclic depsipeptide globomycin, poor stability and production difficulties limit their use in a clinical setting. We harness computational design to generate stable de novo cyclic peptide analogues of globomycin. Only 12 peptides needed to be synthesized and tested to yield potent inhibitors, avoiding costly preparation of large libraries and screening campaigns. The most potent analogues showed comparable or better antimicrobial activity than globomycin in microdilution assays against ESKAPE-E pathogens. This work highlights computational design as a general strategy to combat antibiotic resistance.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
There remains a critical need for new antibiotics against multi-drug-resistant Gram-negative bacteria, a major global threat that continues to impact mortality rates. Lipoprotein signal peptidase II is an essential enzyme in the lipoprotein biosynthetic pathway of Gram-negative bacteria, making it an attractive target for antibacterial drug discovery. Although natural inhibitors of LspA have been identified, such as the cyclic depsipeptide globomycin, poor stability and production difficulties limit their use in a clinical setting. We harness computational design to generate stable de novo cyclic peptide analogues of globomycin. Only 12 peptides needed to be synthesized and tested to yield potent inhibitors, avoiding costly preparation of large libraries and screening campaigns. The most potent analogues showed comparable or better antimicrobial activity than globomycin in microdilution assays against ESKAPE-E pathogens. This work highlights computational design as a general strategy to combat antibiotic resistance.
7.
Sahtoe, Danny D.; Andrzejewska, Ewa A.; Han, Hannah L.; Rennella, Enrico; Schneider, Matthias M.; Meisl, Georg; Ahlrichs, Maggie; Decarreau, Justin; Nguyen, Hannah; Kang, Alex; Levine, Paul; Lamb, Mila; Li, Xinting; Bera, Asim K.; Kay, Lewis E.; Knowles, Tuomas P. J.; Baker, David
Design of amyloidogenic peptide traps Journal Article
In: Nature Chemical Biology, 2024.
@article{Sahtoe2024,
title = {Design of amyloidogenic peptide traps},
author = {Danny D. Sahtoe and Ewa A. Andrzejewska and Hannah L. Han and Enrico Rennella and Matthias M. Schneider and Georg Meisl and Maggie Ahlrichs and Justin Decarreau and Hannah Nguyen and Alex Kang and Paul Levine and Mila Lamb and Xinting Li and Asim K. Bera and Lewis E. Kay and Tuomas P. J. Knowles and David Baker},
url = {https://www.nature.com/articles/s41589-024-01578-5, Nature Chemical Biology [Open Access]},
doi = {10.1038/s41589-024-01578-5},
year = {2024},
date = {2024-03-19},
urldate = {2024-03-19},
journal = {Nature Chemical Biology},
publisher = {Springer Science and Business Media LLC},
abstract = {Segments of proteins with high β-strand propensity can self-associate to form amyloid fibrils implicated in many diseases. We describe a general approach to bind such segments in β-strand and β-hairpin conformations using de novo designed scaffolds that contain deep peptide-binding clefts. The designs bind their cognate peptides in vitro with nanomolar affinities. The crystal structure of a designed protein−peptide complex is close to the design model, and NMR characterization reveals how the peptide-binding cleft is protected in the apo state. We use the approach to design binders to the amyloid-forming proteins transthyretin, tau, serum amyloid A1 and amyloid β1−42 (Aβ42). The Aβ binders block the assembly of Aβ fibrils as effectively as the most potent of the clinically tested antibodies to date and protect cells from toxic Aβ42 species.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Segments of proteins with high β-strand propensity can self-associate to form amyloid fibrils implicated in many diseases. We describe a general approach to bind such segments in β-strand and β-hairpin conformations using de novo designed scaffolds that contain deep peptide-binding clefts. The designs bind their cognate peptides in vitro with nanomolar affinities. The crystal structure of a designed protein−peptide complex is close to the design model, and NMR characterization reveals how the peptide-binding cleft is protected in the apo state. We use the approach to design binders to the amyloid-forming proteins transthyretin, tau, serum amyloid A1 and amyloid β1−42 (Aβ42). The Aβ binders block the assembly of Aβ fibrils as effectively as the most potent of the clinically tested antibodies to date and protect cells from toxic Aβ42 species.
8.
Mateos, Diego Lopez; Murray, Adam M.; Nguyen, Hai M.; Venkatesh, Preetham; Koepnick, Brian; Baker, David; Wulff, Heike; Yarov-Yarovoy, Vladimir
Computational design of binders targeting the VSDIV from NaV1.7 sodium channel Journal Article
In: Biophysical Journal, 2024.
@article{Mateos2024,
title = {Computational design of binders targeting the VSDIV from NaV1.7 sodium channel},
author = {Diego Lopez Mateos and Adam M. Murray and Hai M. Nguyen and Preetham Venkatesh and Brian Koepnick and David Baker and Heike Wulff and Vladimir Yarov-Yarovoy},
url = {https://www.cell.com/biophysj/abstract/S0006-3495(23)01470-4, Biophysical Journal},
doi = {10.1016/j.bpj.2023.11.770},
year = {2024},
date = {2024-02-08},
urldate = {2024-02-00},
journal = {Biophysical Journal},
publisher = {Elsevier BV},
abstract = {Chronic pain affects about 20% of the US population, but safe treatments are limited. There is an urgent need for effective and non-addictive therapies for chronic pan conditions. Voltage-gated sodium (NaV) channel, NaV1.7, is a key player in pain signaling pathway, making it a promising target for novel pain therapeutics. Achieving high subtype selectivity when targeting NaV channels is of primary importance to avoid impairing vital physiological functions mediated by off-target channels. Efforts to selectively target NaV1.7 have been hindered by the difficulties in targeting NaV1.7 over other NaV channel subtypes. Peptidic gating modifier toxins (GMTs), such as Protoxin-II (ProTx2), are promising scaffolds for novel peptide design targeting ion channels with high potency and subtype selectivity. ProTx2 binds to the second and fourth voltage-sensing domains (VSDII and VSDIV) from NaV1.7 with moderate subtype selectivity and can modulate channel activation and inactivation. In this project, we modeled ProTx2 bound to human NaV1.7 VSDIV in an activated state. We used RoseTTAFold Diffusion and Protein MPNN protein design methods to generate protein binders inspired by ProTx2 binding motif with increased predicted binding affinity for human NaV1.7 VSDIV in an activated state. Additionally, we applied these protein design methods to create de novo binders targeting human NaV1.7 VSDIV in an activated state. We anticipate that trapping the VSDIV in an activated conformation will stabilize an inactivated state of the channel, as activation of VSDIV is coupled with channel fast inactivation. Initial electrophysiological screening of our top in silico binders identified promising candidates that inhibited NaV1.7 in the micromolar range. These binders will undergo further testing and optimization against NaV1.7 to create novel molecular tools to study NaV channel activity and effective and safe therapies for chronic pain.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Chronic pain affects about 20% of the US population, but safe treatments are limited. There is an urgent need for effective and non-addictive therapies for chronic pan conditions. Voltage-gated sodium (NaV) channel, NaV1.7, is a key player in pain signaling pathway, making it a promising target for novel pain therapeutics. Achieving high subtype selectivity when targeting NaV channels is of primary importance to avoid impairing vital physiological functions mediated by off-target channels. Efforts to selectively target NaV1.7 have been hindered by the difficulties in targeting NaV1.7 over other NaV channel subtypes. Peptidic gating modifier toxins (GMTs), such as Protoxin-II (ProTx2), are promising scaffolds for novel peptide design targeting ion channels with high potency and subtype selectivity. ProTx2 binds to the second and fourth voltage-sensing domains (VSDII and VSDIV) from NaV1.7 with moderate subtype selectivity and can modulate channel activation and inactivation. In this project, we modeled ProTx2 bound to human NaV1.7 VSDIV in an activated state. We used RoseTTAFold Diffusion and Protein MPNN protein design methods to generate protein binders inspired by ProTx2 binding motif with increased predicted binding affinity for human NaV1.7 VSDIV in an activated state. Additionally, we applied these protein design methods to create de novo binders targeting human NaV1.7 VSDIV in an activated state. We anticipate that trapping the VSDIV in an activated conformation will stabilize an inactivated state of the channel, as activation of VSDIV is coupled with channel fast inactivation. Initial electrophysiological screening of our top in silico binders identified promising candidates that inhibited NaV1.7 in the micromolar range. These binders will undergo further testing and optimization against NaV1.7 to create novel molecular tools to study NaV channel activity and effective and safe therapies for chronic pain.
9.
Torres, Susana Vázquez; Leung, Philip J Y; Venkatesh, Preetham; Lutz, Isaac D; Hink, Fabian; Huynh, Huu-Hien; Becker, Jessica; Yeh, Andy Hsien-Wei; Juergens, David; Bennett, Nathaniel R; Hoofnagle, Andrew N; Huang, Eric; MacCoss, Michael J; Expòsit, Marc; Lee, Gyu Rie; Bera, Asim K; Kang, Alex; Cruz, Joshmyn De La; Levine, Paul M; Li, Xinting; Lamb, Mila; Gerben, Stacey R; Murray, Analisa; Heine, Piper; Korkmaz, Elif Nihal; Nivala, Jeff; Stewart, Lance; Watson, Joseph L; Rogers, Joseph M; Baker, David
De novo design of high-affinity binders of bioactive helical peptides Journal Article
In: Nature, 2023, ISSN: 1476-4687.
@article{pmid38109936,
title = {De novo design of high-affinity binders of bioactive helical peptides},
author = {Susana Vázquez Torres and Philip J Y Leung and Preetham Venkatesh and Isaac D Lutz and Fabian Hink and Huu-Hien Huynh and Jessica Becker and Andy Hsien-Wei Yeh and David Juergens and Nathaniel R Bennett and Andrew N Hoofnagle and Eric Huang and Michael J MacCoss and Marc Expòsit and Gyu Rie Lee and Asim K Bera and Alex Kang and Joshmyn De La Cruz and Paul M Levine and Xinting Li and Mila Lamb and Stacey R Gerben and Analisa Murray and Piper Heine and Elif Nihal Korkmaz and Jeff Nivala and Lance Stewart and Joseph L Watson and Joseph M Rogers and David Baker},
url = {https://www.nature.com/articles/s41586-023-06953-1, Nature [Open Access]},
doi = {10.1038/s41586-023-06953-1},
issn = {1476-4687},
year = {2023},
date = {2023-12-01},
urldate = {2023-12-01},
journal = {Nature},
abstract = {Many peptide hormones form an alpha-helix upon binding their receptors, and sensitive detection methods for them could contribute to better clinical management of disease. De novo protein design can now generate binders with high affinity and specificity to structured proteins. However, the design of interactions between proteins and short peptides with helical propensity is an unmet challenge. Here, we describe parametric generation and deep learning-based methods for designing proteins to address this challenge. We show that by extending RFdiffusion to enable binder design to flexible targets, and to refining input structure models by successive noising and denoising (partial diffusion), picomolar affinity binders can be generated to helical peptide targets both by refining designs generated with other methods, or completely de novo starting from random noise distributions. To our knowledge these are the highest affinity designed binding proteins against any protein or small molecule target generated directly by computation without any experimental optimisation. The RFdiffusion designs enable the enrichment and subsequent detection of parathyroid hormone and glucagon by mass spectrometry, and the construction of bioluminescence-based protein biosensors. The ability to design binders to conformationally variable targets, and to optimise by partial diffusion both natural and designed proteins, should be broadly useful.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Many peptide hormones form an alpha-helix upon binding their receptors, and sensitive detection methods for them could contribute to better clinical management of disease. De novo protein design can now generate binders with high affinity and specificity to structured proteins. However, the design of interactions between proteins and short peptides with helical propensity is an unmet challenge. Here, we describe parametric generation and deep learning-based methods for designing proteins to address this challenge. We show that by extending RFdiffusion to enable binder design to flexible targets, and to refining input structure models by successive noising and denoising (partial diffusion), picomolar affinity binders can be generated to helical peptide targets both by refining designs generated with other methods, or completely de novo starting from random noise distributions. To our knowledge these are the highest affinity designed binding proteins against any protein or small molecule target generated directly by computation without any experimental optimisation. The RFdiffusion designs enable the enrichment and subsequent detection of parathyroid hormone and glucagon by mass spectrometry, and the construction of bioluminescence-based protein biosensors. The ability to design binders to conformationally variable targets, and to optimise by partial diffusion both natural and designed proteins, should be broadly useful.
10.
Muratspahić, Edin; Deibler, Kristine; Han, Jianming; Tomašević, Nataša; Jadhav, Kirtikumar B; Olivé-Marti, Aina-Leonor; Hochrainer, Nadine; Hellinger, Roland; Koehbach, Johannes; Fay, Jonathan F; Rahman, Mohammad Homaidur; Hegazy, Lamees; Craven, Timothy W; Varga, Balazs R; Bhardwaj, Gaurav; Appourchaux, Kevin; Majumdar, Susruta; Muttenthaler, Markus; Hosseinzadeh, Parisa; Craik, David J; Spetea, Mariana; Che, Tao; Baker, David; Gruber, Christian W
Design and structural validation of peptide-drug conjugate ligands of the kappa-opioid receptor Journal Article
In: Nature Communications, 2023.
@article{Muratspahić2023,
title = {Design and structural validation of peptide-drug conjugate ligands of the kappa-opioid receptor},
author = {Edin Muratspahić and Kristine Deibler and Jianming Han and Nataša Tomašević and Kirtikumar B Jadhav and Aina-Leonor Olivé-Marti and Nadine Hochrainer and Roland Hellinger and Johannes Koehbach and Jonathan F Fay and Mohammad Homaidur Rahman and Lamees Hegazy and Timothy W Craven and Balazs R Varga and Gaurav Bhardwaj and Kevin Appourchaux and Susruta Majumdar and Markus Muttenthaler and Parisa Hosseinzadeh and David J Craik and Mariana Spetea and Tao Che and David Baker and Christian W Gruber},
url = {https://www.nature.com/articles/s41467-023-43718-w, Nature Communications [Open Access]},
doi = {10.1038/s41467-023-43718-w},
year = {2023},
date = {2023-12-01},
urldate = {2023-12-01},
journal = {Nature Communications},
abstract = {Despite the increasing number of GPCR structures and recent advances in peptide design, the development of efficient technologies allowing rational design of high-affinity peptide ligands for single GPCRs remains an unmet challenge. Here, we develop a computational approach for designing conjugates of lariat-shaped macrocyclized peptides and a small molecule opioid ligand. We demonstrate its feasibility by discovering chemical scaffolds for the kappa-opioid receptor (KOR) with desired pharmacological activities. The designed De Novo Cyclic Peptide (DNCP)-β-naloxamine (NalA) exhibit in vitro potent mixed KOR agonism/mu-opioid receptor (MOR) antagonism, nanomolar binding affinity, selectivity, and efficacy bias at KOR. Proof-of-concept in vivo efficacy studies demonstrate that DNCP-β-NalA(1) induces a potent KOR-mediated antinociception in male mice. The high-resolution cryo-EM structure (2.6 Å) of the DNCP-β-NalA-KOR-Gi1 complex and molecular dynamics simulations are harnessed to validate the computational design model. This reveals a network of residues in ECL2/3 and TM6/7 controlling the intrinsic efficacy of KOR. In general, our computational de novo platform overcomes extensive lead optimization encountered in ultra-large library docking and virtual small molecule screening campaigns and offers innovation for GPCR ligand discovery. This may drive the development of next-generation therapeutics for medical applications such as pain conditions.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Despite the increasing number of GPCR structures and recent advances in peptide design, the development of efficient technologies allowing rational design of high-affinity peptide ligands for single GPCRs remains an unmet challenge. Here, we develop a computational approach for designing conjugates of lariat-shaped macrocyclized peptides and a small molecule opioid ligand. We demonstrate its feasibility by discovering chemical scaffolds for the kappa-opioid receptor (KOR) with desired pharmacological activities. The designed De Novo Cyclic Peptide (DNCP)-β-naloxamine (NalA) exhibit in vitro potent mixed KOR agonism/mu-opioid receptor (MOR) antagonism, nanomolar binding affinity, selectivity, and efficacy bias at KOR. Proof-of-concept in vivo efficacy studies demonstrate that DNCP-β-NalA(1) induces a potent KOR-mediated antinociception in male mice. The high-resolution cryo-EM structure (2.6 Å) of the DNCP-β-NalA-KOR-Gi1 complex and molecular dynamics simulations are harnessed to validate the computational design model. This reveals a network of residues in ECL2/3 and TM6/7 controlling the intrinsic efficacy of KOR. In general, our computational de novo platform overcomes extensive lead optimization encountered in ultra-large library docking and virtual small molecule screening campaigns and offers innovation for GPCR ligand discovery. This may drive the development of next-generation therapeutics for medical applications such as pain conditions.
11.
Praetorius, Florian; Leung, Philip J. Y.; Tessmer, Maxx H.; Broerman, Adam; Demakis, Cullen; Dishman, Acacia F.; Pillai, Arvind; Idris, Abbas; Juergens, David; Dauparas, Justas; Li, Xinting; Levine, Paul M.; Lamb, Mila; Ballard, Ryanne K.; Gerben, Stacey R.; Nguyen, Hannah; Kang, Alex; Sankaran, Banumathi; Bera, Asim K.; Volkman, Brian F.; Nivala, Jeff; Stoll, Stefan; Baker, David
Design of stimulus-responsive two-state hinge proteins Journal Article
In: Science, 2023.
@article{Praetorius2023,
title = {Design of stimulus-responsive two-state hinge proteins},
author = {Florian Praetorius and Philip J. Y. Leung and Maxx H. Tessmer and Adam Broerman and Cullen Demakis and Acacia F. Dishman and Arvind Pillai and Abbas Idris and David Juergens and Justas Dauparas and Xinting Li and Paul M. Levine and Mila Lamb and Ryanne K. Ballard and Stacey R. Gerben and Hannah Nguyen and Alex Kang and Banumathi Sankaran and Asim K. Bera and Brian F. Volkman and Jeff Nivala and Stefan Stoll and David Baker},
url = {https://www.science.org/stoken/author-tokens/ST-1381/full, Science (Free Access)},
doi = {10.1126/science.adg7731},
year = {2023},
date = {2023-08-17},
urldate = {2023-08-17},
journal = {Science},
abstract = {In nature, proteins that switch between two conformations in response to environmental stimuli structurally transduce biochemical information in a manner analogous to how transistors control information flow in computing devices. Designing proteins with two distinct but fully structured conformations is a challenge for protein design as it requires sculpting an energy landscape with two distinct minima. Here we describe the design of “hinge” proteins that populate one designed state in the absence of ligand and a second designed state in the presence of ligand. X-ray crystallography, electron microscopy, double electron-electron resonance spectroscopy, and binding measurements demonstrate that despite the significant structural differences the two states are designed with atomic level accuracy and that the conformational and binding equilibria are closely coupled. Natural proteins often adopt multiple conformational states, thereby changing their activity or binding partners in response to another protein, small molecule, or other stimulus. It has been difficult to engineer such conformational switching between two folded states in human-designed proteins. Praetorius et al. developed a hinge-like protein by simultaneously considering both desired states in the design process. The successful designs exhibited a large shift in conformation upon binding to a target peptide helix, which could be tailored for specificity. The authors characterized the protein structures, binding kinetics, and conformational equilibrium of the designs. This work provides the groundwork for generating protein switches that respond to biological triggers and can produce conformational changes that modulate protein assemblies. —Michael A. Funk A two-state design of protein switches that couple effector binding to a conformational change is discussed.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In nature, proteins that switch between two conformations in response to environmental stimuli structurally transduce biochemical information in a manner analogous to how transistors control information flow in computing devices. Designing proteins with two distinct but fully structured conformations is a challenge for protein design as it requires sculpting an energy landscape with two distinct minima. Here we describe the design of “hinge” proteins that populate one designed state in the absence of ligand and a second designed state in the presence of ligand. X-ray crystallography, electron microscopy, double electron-electron resonance spectroscopy, and binding measurements demonstrate that despite the significant structural differences the two states are designed with atomic level accuracy and that the conformational and binding equilibria are closely coupled. Natural proteins often adopt multiple conformational states, thereby changing their activity or binding partners in response to another protein, small molecule, or other stimulus. It has been difficult to engineer such conformational switching between two folded states in human-designed proteins. Praetorius et al. developed a hinge-like protein by simultaneously considering both desired states in the design process. The successful designs exhibited a large shift in conformation upon binding to a target peptide helix, which could be tailored for specificity. The authors characterized the protein structures, binding kinetics, and conformational equilibrium of the designs. This work provides the groundwork for generating protein switches that respond to biological triggers and can produce conformational changes that modulate protein assemblies. —Michael A. Funk A two-state design of protein switches that couple effector binding to a conformational change is discussed.
12.
Danny D. Sahtoe Enrico Rennella, David Baker; Kay, Lewis E.
Exploiting conformational dynamics to modulate the function of designed proteins Journal Article
In: Proceedings of the National Academy of Sciences, 2023.
@article{nokey,
title = {Exploiting conformational dynamics to modulate the function of designed proteins},
author = {Enrico Rennella, Danny D. Sahtoe, David Baker, and Lewis E. Kay
},
url = {https://www.pnas.org/doi/10.1073/pnas.2303149120, PNAS},
doi = {10.1073/pnas.2303149120},
year = {2023},
date = {2023-04-24},
journal = {Proceedings of the National Academy of Sciences},
abstract = {With the recent success in calculating protein structures from amino acid sequences using artificial intelligence-based algorithms, an important next step is to decipher how dynamics is encoded by the primary protein sequence so as to better predict function. Such dynamics information is critical for protein design, where strategies could then focus not only on sequences that fold into particular structures that perform a given task, but would also include low-lying excited protein states that could influence the function of the designed protein. Herein, we illustrate the importance of dynamics in modulating the function of C34, a designed α/β protein that captures β-strands of target ligands and is a member of a family of proteins designed to sequester β-strands and β hairpins of aggregation-prone molecules that lead to a variety of pathologies. Using a strategy to “see” regions of apo C34 that are invisible to NMR spectroscopy as a result of pervasive conformational exchange, as well as a mutagenesis approach whereby C34 molecules are stabilized into a single conformer, we determine the structures of the predominant conformations that are sampled by C34 and show that these attenuate the affinity for cognate peptide. Subsequently, the observed motion is exploited to develop an allosterically regulated peptide binder whose binding affinity can be controlled through the addition of a second molecule. Our study emphasizes the unique role that NMR can play in directing the design process and in the construction of new molecules with more complex functionality.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
With the recent success in calculating protein structures from amino acid sequences using artificial intelligence-based algorithms, an important next step is to decipher how dynamics is encoded by the primary protein sequence so as to better predict function. Such dynamics information is critical for protein design, where strategies could then focus not only on sequences that fold into particular structures that perform a given task, but would also include low-lying excited protein states that could influence the function of the designed protein. Herein, we illustrate the importance of dynamics in modulating the function of C34, a designed α/β protein that captures β-strands of target ligands and is a member of a family of proteins designed to sequester β-strands and β hairpins of aggregation-prone molecules that lead to a variety of pathologies. Using a strategy to “see” regions of apo C34 that are invisible to NMR spectroscopy as a result of pervasive conformational exchange, as well as a mutagenesis approach whereby C34 molecules are stabilized into a single conformer, we determine the structures of the predominant conformations that are sampled by C34 and show that these attenuate the affinity for cognate peptide. Subsequently, the observed motion is exploited to develop an allosterically regulated peptide binder whose binding affinity can be controlled through the addition of a second molecule. Our study emphasizes the unique role that NMR can play in directing the design process and in the construction of new molecules with more complex functionality.
13.
Watson, Paris R.; Gupta, Suchetana; Hosseinzadeh, Parisa; Brown, Benjamin P.; Baker, David; Christianson, David W.
Macrocyclic Octapeptide Binding and Inferences on Protein Substrate Binding to Histone Deacetylase 6 Journal Article
In: ACS Chemical Biology, 2023.
@article{Watson0000,
title = {Macrocyclic Octapeptide Binding and Inferences on Protein Substrate Binding to Histone Deacetylase 6},
author = {Watson, Paris R.
and Gupta, Suchetana
and Hosseinzadeh, Parisa
and Brown, Benjamin P.
and Baker, David
and Christianson, David W.},
url = {https://pubs.acs.org/doi/full/10.1021/acschembio.3c00113, ACS Chem. Biol.
https://www.bakerlab.org/wp-content/uploads/2023/04/acschembio.3c00113.pdf, PDF},
doi = {10.1021/acschembio.3c00113},
year = {2023},
date = {2023-04-07},
urldate = {2023-04-07},
journal = {ACS Chemical Biology},
abstract = {Histone deacetylases (HDACs) are essential for the regulation of myriad biological processes, and their aberrant function is implicated in cancer, neurodegeneration, and other diseases. The cytosolic isozyme HDAC6 is unique among the greater family of deacetylases in that it contains two catalytic domains, CD1 and CD2. HDAC6 CD2 is responsible for tubulin deacetylase and tau deacetylase activities, inhibition of which is a key goal as new therapeutic approaches are explored. Of particular interest as HDAC inhibitors are naturally occurring cyclic tetrapeptides such as Trapoxin A or HC Toxin, or the cyclic depsipeptides Largazole and Romidepsin. Even more intriguing are larger, computationally designed macrocyclic peptide inhibitors. Here, we report the 2.0 Å resolution crystal structure of HDAC6 CD2 complexed with macrocyclic octapeptide 1. Comparison with the previously reported structure of the complex with macrocyclic octapeptide 2 reveals that a potent thiolate–zinc interaction made by the unnatural amino acid (S)-2-amino-7-sulfanylheptanoic acid contributes to nanomolar inhibitory potency for each inhibitor. Apart from this zinc-binding residue, octapeptides adopt strikingly different overall conformations and make few direct hydrogen bonds with the protein. Intermolecular interactions are dominated by water-mediated hydrogen bonds; in essence, water molecules appear to cushion the enzyme–octapeptide interface. In view of the broad specificity observed for protein substrates of HDAC6 CD2, we suggest that the binding of macrocyclic octapeptides may mimic certain features of the binding of macromolecular protein substrates.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Histone deacetylases (HDACs) are essential for the regulation of myriad biological processes, and their aberrant function is implicated in cancer, neurodegeneration, and other diseases. The cytosolic isozyme HDAC6 is unique among the greater family of deacetylases in that it contains two catalytic domains, CD1 and CD2. HDAC6 CD2 is responsible for tubulin deacetylase and tau deacetylase activities, inhibition of which is a key goal as new therapeutic approaches are explored. Of particular interest as HDAC inhibitors are naturally occurring cyclic tetrapeptides such as Trapoxin A or HC Toxin, or the cyclic depsipeptides Largazole and Romidepsin. Even more intriguing are larger, computationally designed macrocyclic peptide inhibitors. Here, we report the 2.0 Å resolution crystal structure of HDAC6 CD2 complexed with macrocyclic octapeptide 1. Comparison with the previously reported structure of the complex with macrocyclic octapeptide 2 reveals that a potent thiolate–zinc interaction made by the unnatural amino acid (S)-2-amino-7-sulfanylheptanoic acid contributes to nanomolar inhibitory potency for each inhibitor. Apart from this zinc-binding residue, octapeptides adopt strikingly different overall conformations and make few direct hydrogen bonds with the protein. Intermolecular interactions are dominated by water-mediated hydrogen bonds; in essence, water molecules appear to cushion the enzyme–octapeptide interface. In view of the broad specificity observed for protein substrates of HDAC6 CD2, we suggest that the binding of macrocyclic octapeptides may mimic certain features of the binding of macromolecular protein substrates.
14.
Wu, Kejia; Bai, Hua; Chang, Ya-Ting; Redler, Rachel; McNally, Kerrie E.; Sheffler, William; Brunette, T. J.; Hicks, Derrick R.; Morgan, Tomos E.; Stevens, Tim J.; Broerman, Adam; Goreshnik, Inna; DeWitt, Michelle; Chow, Cameron M.; Shen, Yihang; Stewart, Lance; Derivery, Emmanuel; Silva, Daniel Adriano; Bhabha, Gira; Ekiert, Damian C.; Baker, David
De novo design of modular peptide-binding proteins by superhelical matching Journal Article
In: Nature, 2023.
@article{Wu2023,
title = {De novo design of modular peptide-binding proteins by superhelical matching},
author = {Wu, Kejia
and Bai, Hua
and Chang, Ya-Ting
and Redler, Rachel
and McNally, Kerrie E.
and Sheffler, William
and Brunette, T. J.
and Hicks, Derrick R.
and Morgan, Tomos E.
and Stevens, Tim J.
and Broerman, Adam
and Goreshnik, Inna
and DeWitt, Michelle
and Chow, Cameron M.
and Shen, Yihang
and Stewart, Lance
and Derivery, Emmanuel
and Silva, Daniel Adriano
and Bhabha, Gira
and Ekiert, Damian C.
and Baker, David},
url = {https://www.nature.com/articles/s41586-023-05909-9, Nature (Open-access)},
doi = {10.1038/s41586-023-05909-9},
year = {2023},
date = {2023-04-05},
urldate = {2023-04-05},
journal = {Nature},
abstract = {General approaches for designing sequence-specific peptide-binding proteins would have wide utility in proteomics and synthetic biology. However, designing peptide-binding proteins is challenging, as most peptides do not have defined structures in isolation, and hydrogen bonds must be made to the buried polar groups in the peptide backbone1–3. Here, inspired by natural and re-engineered protein–peptide systems4–11, we set out to design proteins made out of repeating units that bind peptides with repeating sequences, with a one-to-one correspondence between the repeat units of the protein and those of the peptide. We use geometric hashing to identify protein backbones and peptide-docking arrangements that are compatible with bidentate hydrogen bonds between the side chains of the protein and the peptide backbone12. The remainder of the protein sequence is then optimized for folding and peptide binding. We design repeat proteins to bind to six different tripeptide-repeat sequences in polyproline II conformations. The proteins are hyperstable and bind to four to six tandem repeats of their tripeptide targets with nanomolar to picomolar affinities in vitro and in living cells. Crystal structures reveal repeating interactions between protein and peptide interactions as designed, including ladders of hydrogen bonds from protein side chains to peptide backbones. By redesigning the binding interfaces of individual repeat units, specificity can be achieved for non-repeating peptide sequences and for disordered regions of native proteins.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
General approaches for designing sequence-specific peptide-binding proteins would have wide utility in proteomics and synthetic biology. However, designing peptide-binding proteins is challenging, as most peptides do not have defined structures in isolation, and hydrogen bonds must be made to the buried polar groups in the peptide backbone1–3. Here, inspired by natural and re-engineered protein–peptide systems4–11, we set out to design proteins made out of repeating units that bind peptides with repeating sequences, with a one-to-one correspondence between the repeat units of the protein and those of the peptide. We use geometric hashing to identify protein backbones and peptide-docking arrangements that are compatible with bidentate hydrogen bonds between the side chains of the protein and the peptide backbone12. The remainder of the protein sequence is then optimized for folding and peptide binding. We design repeat proteins to bind to six different tripeptide-repeat sequences in polyproline II conformations. The proteins are hyperstable and bind to four to six tandem repeats of their tripeptide targets with nanomolar to picomolar affinities in vitro and in living cells. Crystal structures reveal repeating interactions between protein and peptide interactions as designed, including ladders of hydrogen bonds from protein side chains to peptide backbones. By redesigning the binding interfaces of individual repeat units, specificity can be achieved for non-repeating peptide sequences and for disordered regions of native proteins.
15.
Justas Dauparas Amir Motmaen, Minkyung Baek
Peptide-binding specificity prediction using fine-tuned protein structure prediction networks Journal Article
In: Proceedings of the National Academy of Sciences, 2023.
@article{nokey,
title = {Peptide-binding specificity prediction using fine-tuned protein structure prediction networks},
author = {Amir Motmaen, Justas Dauparas, Minkyung Baek, Mohamad H. Abedi, David Baker, Philip Bradley},
url = {https://www.pnas.org/doi/10.1073/pnas.2216697120, PNAS (Open Access)},
doi = {10.1073/pnas.2216697120},
year = {2023},
date = {2023-02-21},
urldate = {2023-02-21},
journal = {Proceedings of the National Academy of Sciences},
abstract = {Peptide-binding proteins play key roles in biology, and predicting their binding specificity is a long-standing challenge. While considerable protein structural information is available, the most successful current methods use sequence information alone, in part because it has been a challenge to model the subtle structural changes accompanying sequence substitutions. Protein structure prediction networks such as AlphaFold model sequence-structure relationships very accurately, and we reasoned that if it were possible to specifically train such networks on binding data, more generalizable models could be created. We show that placing a classifier on top of the AlphaFold network and fine-tuning the combined network parameters for both classification and structure prediction accuracy leads to a model with strong generalizable performance on a wide range of Class I and Class II peptide-MHC interactions that approaches the overall performance of the state-of-the-art NetMHCpan sequence-based method. The peptide-MHC optimized model shows excellent performance in distinguishing binding and non-binding peptides to SH3 and PDZ domains. This ability to generalize well beyond the training set far exceeds that of sequence-only models and should be particularly powerful for systems where less experimental data are available.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Peptide-binding proteins play key roles in biology, and predicting their binding specificity is a long-standing challenge. While considerable protein structural information is available, the most successful current methods use sequence information alone, in part because it has been a challenge to model the subtle structural changes accompanying sequence substitutions. Protein structure prediction networks such as AlphaFold model sequence-structure relationships very accurately, and we reasoned that if it were possible to specifically train such networks on binding data, more generalizable models could be created. We show that placing a classifier on top of the AlphaFold network and fine-tuning the combined network parameters for both classification and structure prediction accuracy leads to a model with strong generalizable performance on a wide range of Class I and Class II peptide-MHC interactions that approaches the overall performance of the state-of-the-art NetMHCpan sequence-based method. The peptide-MHC optimized model shows excellent performance in distinguishing binding and non-binding peptides to SH3 and PDZ domains. This ability to generalize well beyond the training set far exceeds that of sequence-only models and should be particularly powerful for systems where less experimental data are available.
16.
Praetorius, Florian; Leung, Philip J. Y.; Tessmer, Maxx H.; Broerman, Adam; Demakis, Cullen; Dishman, Acacia F.; Pillai, Arvind; Idris, Abbas; Juergens, David; Dauparas, Justas; Li, Xinting; Levine, Paul M.; Lamb, Mila; Ballard, Ryanne K.; Gerben, Stacey R.; Nguyen, Hannah; Kang, Alex; Sankaran, Banumathi; Bera, Asim K.; Volkman, Brian F.; Nivala, Jeff; Stoll, Stefan; Baker, David
Design of stimulus-responsive two-state hinge proteins Journal Article
In: Science, vol. 381, no. 6659, pp. 754-760, 2023.
@article{doi:10.1126/science.adg7731b,
title = {Design of stimulus-responsive two-state hinge proteins},
author = {Florian Praetorius and Philip J. Y. Leung and Maxx H. Tessmer and Adam Broerman and Cullen Demakis and Acacia F. Dishman and Arvind Pillai and Abbas Idris and David Juergens and Justas Dauparas and Xinting Li and Paul M. Levine and Mila Lamb and Ryanne K. Ballard and Stacey R. Gerben and Hannah Nguyen and Alex Kang and Banumathi Sankaran and Asim K. Bera and Brian F. Volkman and Jeff Nivala and Stefan Stoll and David Baker},
url = {https://www.science.org/doi/abs/10.1126/science.adg7731},
doi = {10.1126/science.adg7731},
year = {2023},
date = {2023-01-01},
journal = {Science},
volume = {381},
number = {6659},
pages = {754-760},
abstract = {In nature, proteins that switch between two conformations in response to environmental stimuli structurally transduce biochemical information in a manner analogous to how transistors control information flow in computing devices. Designing proteins with two distinct but fully structured conformations is a challenge for protein design as it requires sculpting an energy landscape with two distinct minima. Here we describe the design of “hinge” proteins that populate one designed state in the absence of ligand and a second designed state in the presence of ligand. X-ray crystallography, electron microscopy, double electron-electron resonance spectroscopy, and binding measurements demonstrate that despite the significant structural differences the two states are designed with atomic level accuracy and that the conformational and binding equilibria are closely coupled. Natural proteins often adopt multiple conformational states, thereby changing their activity or binding partners in response to another protein, small molecule, or other stimulus. It has been difficult to engineer such conformational switching between two folded states in human-designed proteins. Praetorius et al. developed a hinge-like protein by simultaneously considering both desired states in the design process. The successful designs exhibited a large shift in conformation upon binding to a target peptide helix, which could be tailored for specificity. The authors characterized the protein structures, binding kinetics, and conformational equilibrium of the designs. This work provides the groundwork for generating protein switches that respond to biological triggers and can produce conformational changes that modulate protein assemblies. —Michael A. Funk A two-state design of protein switches that couple effector binding to a conformational change is discussed.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In nature, proteins that switch between two conformations in response to environmental stimuli structurally transduce biochemical information in a manner analogous to how transistors control information flow in computing devices. Designing proteins with two distinct but fully structured conformations is a challenge for protein design as it requires sculpting an energy landscape with two distinct minima. Here we describe the design of “hinge” proteins that populate one designed state in the absence of ligand and a second designed state in the presence of ligand. X-ray crystallography, electron microscopy, double electron-electron resonance spectroscopy, and binding measurements demonstrate that despite the significant structural differences the two states are designed with atomic level accuracy and that the conformational and binding equilibria are closely coupled. Natural proteins often adopt multiple conformational states, thereby changing their activity or binding partners in response to another protein, small molecule, or other stimulus. It has been difficult to engineer such conformational switching between two folded states in human-designed proteins. Praetorius et al. developed a hinge-like protein by simultaneously considering both desired states in the design process. The successful designs exhibited a large shift in conformation upon binding to a target peptide helix, which could be tailored for specificity. The authors characterized the protein structures, binding kinetics, and conformational equilibrium of the designs. This work provides the groundwork for generating protein switches that respond to biological triggers and can produce conformational changes that modulate protein assemblies. —Michael A. Funk A two-state design of protein switches that couple effector binding to a conformational change is discussed.
17.
Said, Meerit Y.; Kang, Christine S.; Wang, Shunzhi; Sheffler, William; Salveson, Patrick J.; Bera, Asim K.; Kang, Alex; Nguyen, Hannah; Ballard, Ryanne; Li, Xinting; Bai, Hua; Stewart, Lance; Levine, Paul; Baker, David
Exploration of Structured Symmetric Cyclic Peptides as Ligands for Metal-Organic Frameworks Journal Article
In: Chemistry of Materials, 2022.
@article{Said2022,
title = {Exploration of Structured Symmetric Cyclic Peptides as Ligands for Metal-Organic Frameworks},
author = {Said, Meerit Y. and Kang, Christine S. and Wang, Shunzhi and Sheffler, William and Salveson, Patrick J. and Bera, Asim K. and Kang, Alex and Nguyen, Hannah and Ballard, Ryanne and Li, Xinting and Bai, Hua and Stewart, Lance and Levine, Paul and Baker, David},
url = {https://pubs.acs.org/doi/10.1021/acs.chemmater.2c02597, Chem. Mater.
https://www.bakerlab.org/wp-content/uploads/2022/10/Said_etal_ChemMater2022_CyclicPeptideMOFs.pdf, PDF},
doi = {/10.1021/acs.chemmater.2c02597},
year = {2022},
date = {2022-10-25},
urldate = {2022-10-25},
journal = {Chemistry of Materials},
abstract = {Despite remarkable advances in the assembly of highly structured coordination polymers and metal–organic frameworks, the rational design of such materials using more conformationally flexible organic ligands such as peptides remains challenging. In an effort to make the design of such materials fully programmable, we first developed a computational design method for generating metal-mediated 3D frameworks using rigid and symmetric peptide macrocycles with metal-coordinating sidechains. We solved the structures of six crystalline networks involving conformationally constrained 6 to 12 residue cyclic peptides with C2, C3, and S2 internal symmetry and three different types of metals (Zn2+, Co2+, or Cu2+) by single-crystal X-ray diffraction, which reveals how the peptide sequences, backbone symmetries, and metal coordination preferences drive the assembly of the resulting structures. In contrast to smaller ligands, these peptides associate through peptide–peptide interactions without full coordination of the metals, contrary to one of the assumptions underlying our computational design method. The cyclic peptides are the largest peptidic ligands reported to form crystalline coordination polymers with transition metals to date, and while more work is required to develop methods for fully programming their crystal structures, the combination of high chemical diversity with synthetic accessibility makes them attractive building blocks for engineering a broader set of new crystalline materials for use in applications such as sensing, asymmetric catalysis, and chiral separation.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Despite remarkable advances in the assembly of highly structured coordination polymers and metal–organic frameworks, the rational design of such materials using more conformationally flexible organic ligands such as peptides remains challenging. In an effort to make the design of such materials fully programmable, we first developed a computational design method for generating metal-mediated 3D frameworks using rigid and symmetric peptide macrocycles with metal-coordinating sidechains. We solved the structures of six crystalline networks involving conformationally constrained 6 to 12 residue cyclic peptides with C2, C3, and S2 internal symmetry and three different types of metals (Zn2+, Co2+, or Cu2+) by single-crystal X-ray diffraction, which reveals how the peptide sequences, backbone symmetries, and metal coordination preferences drive the assembly of the resulting structures. In contrast to smaller ligands, these peptides associate through peptide–peptide interactions without full coordination of the metals, contrary to one of the assumptions underlying our computational design method. The cyclic peptides are the largest peptidic ligands reported to form crystalline coordination polymers with transition metals to date, and while more work is required to develop methods for fully programming their crystal structures, the combination of high chemical diversity with synthetic accessibility makes them attractive building blocks for engineering a broader set of new crystalline materials for use in applications such as sensing, asymmetric catalysis, and chiral separation.
18.
Bhardwaj, Gaurav; O’Connor, Jacob; Rettie, Stephen; Huang, Yen-Hua; Ramelot, Theresa A.; Mulligan, Vikram Khipple; Alpkilic, Gizem Gokce; Palmer, Jonathan; Bera, Asim K.; Bick, Matthew J.; Piazza, Maddalena Di; Li, Xinting; Hosseinzadeh, Parisa; Craven, Timothy W.; Tejero, Roberto; Lauko, Anna; Choi, Ryan; Glynn, Calina; Dong, Linlin; Griffin, Robert; van Voorhis, Wesley C.; Rodriguez, Jose; Stewart, Lance; Montelione, Gaetano T.; Craik, David; Baker, David
Accurate de novo design of membrane-traversing macrocycles Journal Article
In: Cell, 2022.
@article{Bhardwaj2022,
title = {Accurate de novo design of membrane-traversing macrocycles},
author = {Gaurav Bhardwaj and Jacob O’Connor and Stephen Rettie and Yen-Hua Huang and Theresa A. Ramelot and Vikram Khipple Mulligan and Gizem Gokce Alpkilic and Jonathan Palmer and Asim K. Bera and Matthew J. Bick and Maddalena {Di Piazza} and Xinting Li and Parisa Hosseinzadeh and Timothy W. Craven and Roberto Tejero and Anna Lauko and Ryan Choi and Calina Glynn and Linlin Dong and Robert Griffin and Wesley C. {van Voorhis} and Jose Rodriguez and Lance Stewart and Gaetano T. Montelione and David Craik and David Baker},
url = {https://www.sciencedirect.com/science/article/pii/S0092867422009229?via%3Dihub, Cell
https://www.bakerlab.org/wp-content/uploads/2022/08/1-s2.0-S0092867422009229-main.pdf, PDF},
doi = {10.1016/j.cell.2022.07.019},
year = {2022},
date = {2022-08-29},
urldate = {2022-08-29},
journal = {Cell},
abstract = {We use computational design coupled with experimental characterization to systematically investigate the design principles for macrocycle membrane permeability and oral bioavailability. We designed 184 6–12 residue macrocycles with a wide range of predicted structures containing noncanonical backbone modifications and experimentally determined structures of 35; 29 are very close to the computational models. With such control, we show that membrane permeability can be systematically achieved by ensuring all amide (NH) groups are engaged in internal hydrogen bonding interactions. 84 designs over the 6–12 residue size range cross membranes with an apparent permeability greater than 1 × 10−6 cm/s. Designs with exposed NH groups can be made membrane permeable through the design of an alternative isoenergetic fully hydrogen-bonded state favored in the lipid membrane. The ability to robustly design membrane-permeable and orally bioavailable peptides with high structural accuracy should contribute to the next generation of designed macrocycle therapeutics.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We use computational design coupled with experimental characterization to systematically investigate the design principles for macrocycle membrane permeability and oral bioavailability. We designed 184 6–12 residue macrocycles with a wide range of predicted structures containing noncanonical backbone modifications and experimentally determined structures of 35; 29 are very close to the computational models. With such control, we show that membrane permeability can be systematically achieved by ensuring all amide (NH) groups are engaged in internal hydrogen bonding interactions. 84 designs over the 6–12 residue size range cross membranes with an apparent permeability greater than 1 × 10−6 cm/s. Designs with exposed NH groups can be made membrane permeable through the design of an alternative isoenergetic fully hydrogen-bonded state favored in the lipid membrane. The ability to robustly design membrane-permeable and orally bioavailable peptides with high structural accuracy should contribute to the next generation of designed macrocycle therapeutics.
19.
Macé, Kévin; Vadakkepat, Abhinav K.; Redzej, Adam; Lukoyanova, Natalya; Oomen, Clasien; Braun, Nathalie; Ukleja, Marta; Lu, Fang; Costa, Tiago R. D.; Orlova, Elena V.; Baker, David; Cong, Qian; Waksman, Gabriel
Cryo-EM structure of a type IV secretion system Journal Article
In: Nature, 2022.
@article{Macé2022,
title = {Cryo-EM structure of a type IV secretion system},
author = {Macé, Kévin
and Vadakkepat, Abhinav K.
and Redzej, Adam
and Lukoyanova, Natalya
and Oomen, Clasien
and Braun, Nathalie
and Ukleja, Marta
and Lu, Fang
and Costa, Tiago R. D.
and Orlova, Elena V.
and Baker, David
and Cong, Qian
and Waksman, Gabriel},
url = {https://www.nature.com/articles/s41586-022-04859-y, Nature
https://www.bakerlab.org/wp-content/uploads/2022/08/Mace2022s41586-022-04859-y.pdf, PDF},
doi = {10.1038/s41586-022-04859-y},
year = {2022},
date = {2022-07-01},
urldate = {2022-07-01},
journal = {Nature},
abstract = {Bacterial conjugation is the fundamental process of unidirectional transfer of DNAs, often plasmid DNAs, from a donor cell to a recipient cell1. It is the primary means by which antibiotic resistance genes spread among bacterial populations2,3. In Gram-negative bacteria, conjugation is mediated by a large transport apparatus—the conjugative type IV secretion system (T4SS)—produced by the donor cell and embedded in both its outer and inner membranes. The T4SS also elaborates a long extracellular filament—the conjugative pilus—that is essential for DNA transfer4,5. Here we present a high-resolution cryo-electron microscopy (cryo-EM) structure of a 2.8 megadalton T4SS complex composed of 92 polypeptides representing 8 of the 10 essential T4SS components involved in pilus biogenesis. We added the two remaining components to the structural model using co-evolution analysis of protein interfaces, to enable the reconstitution of the entire system including the pilus. This structure describes the exceptionally large protein–protein interaction network required to assemble the many components that constitute a T4SS and provides insights on the unique mechanism by which they elaborate pili.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Bacterial conjugation is the fundamental process of unidirectional transfer of DNAs, often plasmid DNAs, from a donor cell to a recipient cell1. It is the primary means by which antibiotic resistance genes spread among bacterial populations2,3. In Gram-negative bacteria, conjugation is mediated by a large transport apparatus—the conjugative type IV secretion system (T4SS)—produced by the donor cell and embedded in both its outer and inner membranes. The T4SS also elaborates a long extracellular filament—the conjugative pilus—that is essential for DNA transfer4,5. Here we present a high-resolution cryo-electron microscopy (cryo-EM) structure of a 2.8 megadalton T4SS complex composed of 92 polypeptides representing 8 of the 10 essential T4SS components involved in pilus biogenesis. We added the two remaining components to the structural model using co-evolution analysis of protein interfaces, to enable the reconstitution of the entire system including the pilus. This structure describes the exceptionally large protein–protein interaction network required to assemble the many components that constitute a T4SS and provides insights on the unique mechanism by which they elaborate pili.
20.
Levine, Paul M.; Craven, Timothy W.; Li, Xinting; Balana, Aaron T.; Bird, Gregory H.; Godes, Marina; Salveson, Patrick J.; Erickson, Patrick W.; Lamb, Mila; Ahlrichs, Maggie; Murphy, Michael; Ogohara, Cassandra; Said, Meerit Y.; Walensky, Loren D.; Pratt, Matthew R.; Baker, David
Generation of Potent and Stable GLP-1 Analogues Via “Serine Ligation” Journal Article
In: ACS Chemical Biology, 2022.
@article{nokey,
title = {Generation of Potent and Stable GLP-1 Analogues Via “Serine Ligation”},
author = {Levine, Paul M. and Craven, Timothy W. and Li, Xinting and Balana, Aaron T. and Bird, Gregory H. and Godes, Marina and Salveson, Patrick J. and Erickson, Patrick W. and Lamb, Mila and Ahlrichs, Maggie and Murphy, Michael and Ogohara, Cassandra and Said, Meerit Y. and Walensky, Loren D. and Pratt, Matthew R. and Baker, David},
url = {https://pubs.acs.org/doi/abs/10.1021/acschembio.2c00075, ACS Chemical Biology
https://www.bakerlab.org/wp-content/uploads/2022/03/Levine_etal_ACSChemBio2022_GLP-1_ananlogues_by_serine_ligation.pdf, Download PDF},
doi = {10.1021/acschembio.2c00075},
year = {2022},
date = {2022-03-23},
journal = {ACS Chemical Biology},
abstract = {Peptide and protein bioconjugation technologies have revolutionized our ability to site-specifically or chemoselectively install a variety of functional groups for applications in chemical biology and medicine, including the enhancement of bioavailability. Here, we introduce a site-specific bioconjugation strategy inspired by chemical ligation at serine that relies on a noncanonical amino acid containing a 1-amino-2-hydroxy functional group and a salicylaldehyde ester. More specifically, we harness this technology to generate analogues of glucagon-like peptide-1 that resemble Semaglutide, a long-lasting blockbuster drug currently used in the clinic to regulate glucose levels in the blood. We identify peptides that are more potent than unmodified peptide and equipotent to Semaglutide in a cell-based activation assay, improve the stability in human serum, and increase glucose disposal efficiency in vivo. This approach demonstrates the potential of “serine ligation” for various applications in chemical biology, with a particular focus on generating stabilized peptide therapeutics.
},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Peptide and protein bioconjugation technologies have revolutionized our ability to site-specifically or chemoselectively install a variety of functional groups for applications in chemical biology and medicine, including the enhancement of bioavailability. Here, we introduce a site-specific bioconjugation strategy inspired by chemical ligation at serine that relies on a noncanonical amino acid containing a 1-amino-2-hydroxy functional group and a salicylaldehyde ester. More specifically, we harness this technology to generate analogues of glucagon-like peptide-1 that resemble Semaglutide, a long-lasting blockbuster drug currently used in the clinic to regulate glucose levels in the blood. We identify peptides that are more potent than unmodified peptide and equipotent to Semaglutide in a cell-based activation assay, improve the stability in human serum, and increase glucose disposal efficiency in vivo. This approach demonstrates the potential of “serine ligation” for various applications in chemical biology, with a particular focus on generating stabilized peptide therapeutics.
2024
FROM THE LAB
Sidney Lyayuga Lisanza, Jacob Merle Gershon, Samuel W. K. Tipps, Jeremiah Nelson Sims, Lucas Arnoldt, Samuel J. Hendel, Miriam K. Simma, Ge Liu, Muna Yase, Hongwei Wu, Claire D. Tharp, Xinting Li, Alex Kang, Evans Brackenbrough, Asim K. Bera, Stacey Gerben, Bruce J. Wittmann, Andrew C. McShan, David Baker
Multistate and functional protein design using RoseTTAFold sequence space diffusion Journal Article
In: Nature Biotechnology, 2024.
@article{Lisanza2024,
title = {Multistate and functional protein design using RoseTTAFold sequence space diffusion},
author = {Sidney Lyayuga Lisanza and Jacob Merle Gershon and Samuel W. K. Tipps and Jeremiah Nelson Sims and Lucas Arnoldt and Samuel J. Hendel and Miriam K. Simma and Ge Liu and Muna Yase and Hongwei Wu and Claire D. Tharp and Xinting Li and Alex Kang and Evans Brackenbrough and Asim K. Bera and Stacey Gerben and Bruce J. Wittmann and Andrew C. McShan and David Baker},
url = {https://www.nature.com/articles/s41587-024-02395-w, Nature Biotechnology [Open Access]},
doi = {10.1038/s41587-024-02395-w},
year = {2024},
date = {2024-09-25},
urldate = {2024-09-25},
journal = {Nature Biotechnology},
publisher = {Springer Science and Business Media LLC},
abstract = {Protein denoising diffusion probabilistic models are used for the de novo generation of protein backbones but are limited in their ability to guide generation of proteins with sequence-specific attributes and functional properties. To overcome this limitation, we developed ProteinGenerator (PG), a sequence space diffusion model based on RoseTTAFold that simultaneously generates protein sequences and structures. Beginning from a noised sequence representation, PG generates sequence and structure pairs by iterative denoising, guided by desired sequence and structural protein attributes. We designed thermostable proteins with varying amino acid compositions and internal sequence repeats and cage bioactive peptides, such as melittin. By averaging sequence logits between diffusion trajectories with distinct structural constraints, we designed multistate parent–child protein triples in which the same sequence folds to different supersecondary structures when intact in the parent versus split into two child domains. PG design trajectories can be guided by experimental sequence–activity data, providing a general approach for integrated computational and experimental optimization of protein function.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Protein denoising diffusion probabilistic models are used for the de novo generation of protein backbones but are limited in their ability to guide generation of proteins with sequence-specific attributes and functional properties. To overcome this limitation, we developed ProteinGenerator (PG), a sequence space diffusion model based on RoseTTAFold that simultaneously generates protein sequences and structures. Beginning from a noised sequence representation, PG generates sequence and structure pairs by iterative denoising, guided by desired sequence and structural protein attributes. We designed thermostable proteins with varying amino acid compositions and internal sequence repeats and cage bioactive peptides, such as melittin. By averaging sequence logits between diffusion trajectories with distinct structural constraints, we designed multistate parent–child protein triples in which the same sequence folds to different supersecondary structures when intact in the parent versus split into two child domains. PG design trajectories can be guided by experimental sequence–activity data, providing a general approach for integrated computational and experimental optimization of protein function.
Adam P. Moyer, Theresa A. Ramelot, Mariano Curti, Margaret A. Eastman, Alex Kang, Asim K. Bera, Roberto Tejero, Patrick J. Salveson, Carles Curutchet, Elisabet Romero, Gaetano T. Montelione, David Baker
Enumerative Discovery of Noncanonical Polypeptide Secondary Structures Journal Article
In: Journal of the American Chemical Society, 2024.
@article{Moyer2024,
title = {Enumerative Discovery of Noncanonical Polypeptide Secondary Structures},
author = {Adam P. Moyer and Theresa A. Ramelot and Mariano Curti and Margaret A. Eastman and Alex Kang and Asim K. Bera and Roberto Tejero and Patrick J. Salveson and Carles Curutchet and Elisabet Romero and Gaetano T. Montelione and David Baker},
url = {https://pubs.acs.org/doi/full/10.1021/jacs.4c04991, LACS [Open Access]},
doi = {10.1021/jacs.4c04991},
year = {2024},
date = {2024-09-18},
urldate = {2024-09-18},
journal = {Journal of the American Chemical Society},
publisher = {American Chemical Society (ACS)},
abstract = {Energetically favorable local interactions can overcome the entropic cost of chain ordering and cause otherwise flexible polymers to adopt regularly repeating backbone conformations. A prominent example is the α helix present in many protein structures, which is stabilized by i, i + 4 hydrogen bonds between backbone peptide units. With the increased chemical diversity offered by unnatural amino acids and backbones, it has been possible to identify regularly repeating structures not present in proteins, but to date, there has been no systematic approach for identifying new polymers likely to have such structures despite their considerable potential for molecular engineering. Here we describe a systematic approach to search through dipeptide combinations of 130 chemically diverse amino acids to identify those predicted to populate unique low-energy states. We characterize ten newly identified dipeptide repeating structures using circular dichroism spectroscopy and comparison with calculated spectra. NMR and X-ray crystallographic structures of two of these dipeptide-repeat polymers are similar to the computational models. Our approach is readily generalizable to identify low-energy repeating structures for a wide variety of polymers, and our ordered dipeptide repeats provide new building blocks for molecular engineering.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Energetically favorable local interactions can overcome the entropic cost of chain ordering and cause otherwise flexible polymers to adopt regularly repeating backbone conformations. A prominent example is the α helix present in many protein structures, which is stabilized by i, i + 4 hydrogen bonds between backbone peptide units. With the increased chemical diversity offered by unnatural amino acids and backbones, it has been possible to identify regularly repeating structures not present in proteins, but to date, there has been no systematic approach for identifying new polymers likely to have such structures despite their considerable potential for molecular engineering. Here we describe a systematic approach to search through dipeptide combinations of 130 chemically diverse amino acids to identify those predicted to populate unique low-energy states. We characterize ten newly identified dipeptide repeating structures using circular dichroism spectroscopy and comparison with calculated spectra. NMR and X-ray crystallographic structures of two of these dipeptide-repeat polymers are similar to the computational models. Our approach is readily generalizable to identify low-energy repeating structures for a wide variety of polymers, and our ordered dipeptide repeats provide new building blocks for molecular engineering.
Arvind Pillai, Abbas Idris, Annika Philomin, Connor Weidle, Rebecca Skotheim, Philip J. Y. Leung, Adam Broerman, Cullen Demakis, Andrew J. Borst, Florian Praetorius, David Baker
De novo design of allosterically switchable protein assemblies Journal Article
In: Nature, 2024.
@article{Pillai2024,
title = {De novo design of allosterically switchable protein assemblies},
author = {Arvind Pillai and Abbas Idris and Annika Philomin and Connor Weidle and Rebecca Skotheim and Philip J. Y. Leung and Adam Broerman and Cullen Demakis and Andrew J. Borst and Florian Praetorius and David Baker},
url = {https://www.nature.com/articles/s41586-024-07813-2, Nature [Open Access]},
doi = {10.1038/s41586-024-07813-2},
year = {2024},
date = {2024-08-14},
urldate = {2024-08-14},
journal = {Nature},
publisher = {Springer Science and Business Media LLC},
abstract = {Allosteric modulation of protein function, wherein the binding of an effector to a protein triggers conformational changes at distant functional sites, plays a central part in the control of metabolism and cell signalling. There has been considerable interest in designing allosteric systems, both to gain insight into the mechanisms underlying such ‘action at a distance’ modulation and to create synthetic proteins whose functions can be regulated by effectors. However, emulating the subtle conformational changes distributed across many residues, characteristic of natural allosteric proteins, is a significant challenge. Here, inspired by the classic Monod–Wyman–Changeux model of cooperativity, we investigate the de novo design of allostery through rigid-body coupling of peptide-switchable hinge modules to protein interfaces that direct the formation of alternative oligomeric states. We find that this approach can be used to generate a wide variety of allosterically switchable systems, including cyclic rings that incorporate or eject subunits in response to peptide binding and dihedral cages that undergo effector-induced disassembly. Size-exclusion chromatography, mass photometry and electron microscopy reveal that these designed allosteric protein assemblies closely resemble the design models in both the presence and absence of peptide effectors and can have ligand-binding cooperativity comparable to classic natural systems such as haemoglobin. Our results indicate that allostery can arise from global coupling of the energetics of protein substructures without optimized side-chain–side-chain allosteric communication pathways and provide a roadmap for generating allosterically triggerable delivery systems, protein nanomachines and cellular feedback control circuitry.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Allosteric modulation of protein function, wherein the binding of an effector to a protein triggers conformational changes at distant functional sites, plays a central part in the control of metabolism and cell signalling. There has been considerable interest in designing allosteric systems, both to gain insight into the mechanisms underlying such ‘action at a distance’ modulation and to create synthetic proteins whose functions can be regulated by effectors. However, emulating the subtle conformational changes distributed across many residues, characteristic of natural allosteric proteins, is a significant challenge. Here, inspired by the classic Monod–Wyman–Changeux model of cooperativity, we investigate the de novo design of allostery through rigid-body coupling of peptide-switchable hinge modules to protein interfaces that direct the formation of alternative oligomeric states. We find that this approach can be used to generate a wide variety of allosterically switchable systems, including cyclic rings that incorporate or eject subunits in response to peptide binding and dihedral cages that undergo effector-induced disassembly. Size-exclusion chromatography, mass photometry and electron microscopy reveal that these designed allosteric protein assemblies closely resemble the design models in both the presence and absence of peptide effectors and can have ligand-binding cooperativity comparable to classic natural systems such as haemoglobin. Our results indicate that allostery can arise from global coupling of the energetics of protein substructures without optimized side-chain–side-chain allosteric communication pathways and provide a roadmap for generating allosterically triggerable delivery systems, protein nanomachines and cellular feedback control circuitry.
Jiang, Hanlun and Jude, Kevin M. and Wu, Kejia and Fallas, Jorge and Ueda, George and Brunette, T. J. and Hicks, Derrick R. and Pyles, Harley and Yang, Aerin and Carter, Lauren and Lamb, Mila and Li, Xinting and Levine, Paul M. and Stewart, Lance and Garcia, K. Christopher and Baker, David
De novo design of buttressed loops for sculpting protein functions Journal Article
In: Nature Chemical Biology, 2024.
@article{Jiang2024,
title = {De novo design of buttressed loops for sculpting protein functions},
author = {Jiang, Hanlun
and Jude, Kevin M.
and Wu, Kejia
and Fallas, Jorge
and Ueda, George
and Brunette, T. J.
and Hicks, Derrick R.
and Pyles, Harley
and Yang, Aerin
and Carter, Lauren
and Lamb, Mila
and Li, Xinting
and Levine, Paul M.
and Stewart, Lance
and Garcia, K. Christopher
and Baker, David},
url = {https://www.nature.com/articles/s41589-024-01632-2, Nature Chemical Biology [Open Access]
https://www.bakerlab.org/wp-content/uploads/2024/05/s41589-024-01632-2.pdf, PDF},
doi = {10.1038/s41589-024-01632-2},
year = {2024},
date = {2024-05-30},
urldate = {2024-05-30},
journal = {Nature Chemical Biology},
abstract = {In natural proteins, structured loops have central roles in molecular recognition, signal transduction and enzyme catalysis. However, because of the intrinsic flexibility and irregularity of loop regions, organizing multiple structured loops at protein functional sites has been very difficult to achieve by de novo protein design. Here we describe a solution to this problem that designs tandem repeat proteins with structured loops (9–14 residues) buttressed by extensive hydrogen bonding interactions. Experimental characterization shows that the designs are monodisperse, highly soluble, folded and thermally stable. Crystal structures are in close agreement with the design models, with the loops structured and buttressed as designed. We demonstrate the functionality afforded by loop buttressing by designing and characterizing binders for extended peptides in which the loops form one side of an extended binding pocket. The ability to design multiple structured loops should contribute generally to efforts to design new protein functions.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In natural proteins, structured loops have central roles in molecular recognition, signal transduction and enzyme catalysis. However, because of the intrinsic flexibility and irregularity of loop regions, organizing multiple structured loops at protein functional sites has been very difficult to achieve by de novo protein design. Here we describe a solution to this problem that designs tandem repeat proteins with structured loops (9–14 residues) buttressed by extensive hydrogen bonding interactions. Experimental characterization shows that the designs are monodisperse, highly soluble, folded and thermally stable. Crystal structures are in close agreement with the design models, with the loops structured and buttressed as designed. We demonstrate the functionality afforded by loop buttressing by designing and characterizing binders for extended peptides in which the loops form one side of an extended binding pocket. The ability to design multiple structured loops should contribute generally to efforts to design new protein functions.
Timothy W. Craven, Mark D. Nolan, Jonathan Bailey, Samir Olatunji, Samantha J. Bann, Katherine Bowen, Nikita Ostrovitsa, Thaina M. Da Costa, Ross D. Ballantine, Dietmar Weichert, Paul M. Levine, Lance J. Stewart, Gaurav Bhardwaj, Joan A. Geoghegan, Stephen A. Cochrane, Eoin M. Scanlan, Martin Caffrey, David Baker
Computational Design of Cyclic Peptide Inhibitors of a Bacterial Membrane Lipoprotein Peptidase Journal Article
In: ACS Chemical Biology, 2024.
@article{Craven2024,
title = {Computational Design of Cyclic Peptide Inhibitors of a Bacterial Membrane Lipoprotein Peptidase},
author = {Timothy W. Craven and Mark D. Nolan and Jonathan Bailey and Samir Olatunji and Samantha J. Bann and Katherine Bowen and Nikita Ostrovitsa and Thaina M. Da Costa and Ross D. Ballantine and Dietmar Weichert and Paul M. Levine and Lance J. Stewart and Gaurav Bhardwaj and Joan A. Geoghegan and Stephen A. Cochrane and Eoin M. Scanlan and Martin Caffrey and David Baker},
url = {https://pubs.acs.org/doi/10.1021/acschembio.4c00076, ACS Chem. Bio. [Open Access]
https://www.bakerlab.org/wp-content/uploads/2024/05/craven-et-al-2024-computational-design-of-cyclic-peptide-inhibitors-of-a-bacterial-membrane-lipoprotein-peptidase.pdf, PDF},
doi = {10.1021/acschembio.4c00076},
year = {2024},
date = {2024-05-07},
urldate = {2024-05-07},
journal = {ACS Chemical Biology},
publisher = {American Chemical Society (ACS)},
abstract = {There remains a critical need for new antibiotics against multi-drug-resistant Gram-negative bacteria, a major global threat that continues to impact mortality rates. Lipoprotein signal peptidase II is an essential enzyme in the lipoprotein biosynthetic pathway of Gram-negative bacteria, making it an attractive target for antibacterial drug discovery. Although natural inhibitors of LspA have been identified, such as the cyclic depsipeptide globomycin, poor stability and production difficulties limit their use in a clinical setting. We harness computational design to generate stable de novo cyclic peptide analogues of globomycin. Only 12 peptides needed to be synthesized and tested to yield potent inhibitors, avoiding costly preparation of large libraries and screening campaigns. The most potent analogues showed comparable or better antimicrobial activity than globomycin in microdilution assays against ESKAPE-E pathogens. This work highlights computational design as a general strategy to combat antibiotic resistance.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
There remains a critical need for new antibiotics against multi-drug-resistant Gram-negative bacteria, a major global threat that continues to impact mortality rates. Lipoprotein signal peptidase II is an essential enzyme in the lipoprotein biosynthetic pathway of Gram-negative bacteria, making it an attractive target for antibacterial drug discovery. Although natural inhibitors of LspA have been identified, such as the cyclic depsipeptide globomycin, poor stability and production difficulties limit their use in a clinical setting. We harness computational design to generate stable de novo cyclic peptide analogues of globomycin. Only 12 peptides needed to be synthesized and tested to yield potent inhibitors, avoiding costly preparation of large libraries and screening campaigns. The most potent analogues showed comparable or better antimicrobial activity than globomycin in microdilution assays against ESKAPE-E pathogens. This work highlights computational design as a general strategy to combat antibiotic resistance.
Danny D. Sahtoe, Ewa A. Andrzejewska, Hannah L. Han, Enrico Rennella, Matthias M. Schneider, Georg Meisl, Maggie Ahlrichs, Justin Decarreau, Hannah Nguyen, Alex Kang, Paul Levine, Mila Lamb, Xinting Li, Asim K. Bera, Lewis E. Kay, Tuomas P. J. Knowles, David Baker
Design of amyloidogenic peptide traps Journal Article
In: Nature Chemical Biology, 2024.
@article{Sahtoe2024,
title = {Design of amyloidogenic peptide traps},
author = {Danny D. Sahtoe and Ewa A. Andrzejewska and Hannah L. Han and Enrico Rennella and Matthias M. Schneider and Georg Meisl and Maggie Ahlrichs and Justin Decarreau and Hannah Nguyen and Alex Kang and Paul Levine and Mila Lamb and Xinting Li and Asim K. Bera and Lewis E. Kay and Tuomas P. J. Knowles and David Baker},
url = {https://www.nature.com/articles/s41589-024-01578-5, Nature Chemical Biology [Open Access]},
doi = {10.1038/s41589-024-01578-5},
year = {2024},
date = {2024-03-19},
urldate = {2024-03-19},
journal = {Nature Chemical Biology},
publisher = {Springer Science and Business Media LLC},
abstract = {Segments of proteins with high β-strand propensity can self-associate to form amyloid fibrils implicated in many diseases. We describe a general approach to bind such segments in β-strand and β-hairpin conformations using de novo designed scaffolds that contain deep peptide-binding clefts. The designs bind their cognate peptides in vitro with nanomolar affinities. The crystal structure of a designed protein−peptide complex is close to the design model, and NMR characterization reveals how the peptide-binding cleft is protected in the apo state. We use the approach to design binders to the amyloid-forming proteins transthyretin, tau, serum amyloid A1 and amyloid β1−42 (Aβ42). The Aβ binders block the assembly of Aβ fibrils as effectively as the most potent of the clinically tested antibodies to date and protect cells from toxic Aβ42 species.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Segments of proteins with high β-strand propensity can self-associate to form amyloid fibrils implicated in many diseases. We describe a general approach to bind such segments in β-strand and β-hairpin conformations using de novo designed scaffolds that contain deep peptide-binding clefts. The designs bind their cognate peptides in vitro with nanomolar affinities. The crystal structure of a designed protein−peptide complex is close to the design model, and NMR characterization reveals how the peptide-binding cleft is protected in the apo state. We use the approach to design binders to the amyloid-forming proteins transthyretin, tau, serum amyloid A1 and amyloid β1−42 (Aβ42). The Aβ binders block the assembly of Aβ fibrils as effectively as the most potent of the clinically tested antibodies to date and protect cells from toxic Aβ42 species.
COLLABORATOR LED
Ke Sun, Sicong Li, Bowen Zheng, Yanlei Zhu, Tongyue Wang, Mingfu Liang, Yue Yao, Kairan Zhang, Jizhong Zhang, Hongyong Li, Dongyang Han, Jishen Zheng, Brian Coventry, Longxing Cao, David Baker, Lei Liu, Peilong Lu
Accurate de novo design of heterochiral protein–protein interactions Journal Article
In: Cell Research, 2024.
@article{Sun2024,
title = {Accurate de novo design of heterochiral protein–protein interactions},
author = {Ke Sun and Sicong Li and Bowen Zheng and Yanlei Zhu and Tongyue Wang and Mingfu Liang and Yue Yao and Kairan Zhang and Jizhong Zhang and Hongyong Li and Dongyang Han and Jishen Zheng and Brian Coventry and Longxing Cao and David Baker and Lei Liu and Peilong Lu},
url = {https://www.nature.com/articles/s41422-024-01014-2, Cell Research [Open Access]},
doi = {10.1038/s41422-024-01014-2},
year = {2024},
date = {2024-08-14},
urldate = {2024-08-14},
journal = {Cell Research},
publisher = {Springer Science and Business Media LLC},
abstract = {Abiotic d-proteins that selectively bind to natural l-proteins have gained significant biotechnological interest. However, the underlying structural principles governing such heterochiral protein–protein interactions remain largely unknown. In this study, we present the de novo design of d-proteins consisting of 50–65 residues, aiming to target specific surface regions of l-proteins or l-peptides. Our designer d-protein binders exhibit nanomolar affinity toward an artificial l-peptide, as well as two naturally occurring proteins of therapeutic significance: the D5 domain of human tropomyosin receptor kinase A (TrkA) and human interleukin-6 (IL-6). Notably, these d-protein binders demonstrate high enantiomeric specificity and target specificity. In cell-based experiments, designer d-protein binders effectively inhibited the downstream signaling of TrkA and IL-6 with high potency. Moreover, these binders exhibited remarkable thermal stability and resistance to protease degradation. Crystal structure of the designed heterochiral d-protein–l-peptide complex, obtained at a resolution of 2.0 Å, closely resembled the design model, indicating that the computational method employed is highly accurate. Furthermore, the crystal structure provides valuable information regarding the interactions between helical l-peptides and d-proteins, particularly elucidating a novel mode of heterochiral helix–helix interactions. Leveraging the design of d-proteins specifically targeting l-peptides or l-proteins opens up avenues for systematic exploration of the mirror-image protein universe, paving the way for a diverse range of applications.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Abiotic d-proteins that selectively bind to natural l-proteins have gained significant biotechnological interest. However, the underlying structural principles governing such heterochiral protein–protein interactions remain largely unknown. In this study, we present the de novo design of d-proteins consisting of 50–65 residues, aiming to target specific surface regions of l-proteins or l-peptides. Our designer d-protein binders exhibit nanomolar affinity toward an artificial l-peptide, as well as two naturally occurring proteins of therapeutic significance: the D5 domain of human tropomyosin receptor kinase A (TrkA) and human interleukin-6 (IL-6). Notably, these d-protein binders demonstrate high enantiomeric specificity and target specificity. In cell-based experiments, designer d-protein binders effectively inhibited the downstream signaling of TrkA and IL-6 with high potency. Moreover, these binders exhibited remarkable thermal stability and resistance to protease degradation. Crystal structure of the designed heterochiral d-protein–l-peptide complex, obtained at a resolution of 2.0 Å, closely resembled the design model, indicating that the computational method employed is highly accurate. Furthermore, the crystal structure provides valuable information regarding the interactions between helical l-peptides and d-proteins, particularly elucidating a novel mode of heterochiral helix–helix interactions. Leveraging the design of d-proteins specifically targeting l-peptides or l-proteins opens up avenues for systematic exploration of the mirror-image protein universe, paving the way for a diverse range of applications.
Diego Lopez Mateos, Adam M. Murray, Hai M. Nguyen, Preetham Venkatesh, Brian Koepnick, David Baker, Heike Wulff, Vladimir Yarov-Yarovoy
Computational design of binders targeting the VSDIV from NaV1.7 sodium channel Journal Article
In: Biophysical Journal, 2024.
@article{Mateos2024,
title = {Computational design of binders targeting the VSDIV from NaV1.7 sodium channel},
author = {Diego Lopez Mateos and Adam M. Murray and Hai M. Nguyen and Preetham Venkatesh and Brian Koepnick and David Baker and Heike Wulff and Vladimir Yarov-Yarovoy},
url = {https://www.cell.com/biophysj/abstract/S0006-3495(23)01470-4, Biophysical Journal},
doi = {10.1016/j.bpj.2023.11.770},
year = {2024},
date = {2024-02-08},
urldate = {2024-02-00},
journal = {Biophysical Journal},
publisher = {Elsevier BV},
abstract = {Chronic pain affects about 20% of the US population, but safe treatments are limited. There is an urgent need for effective and non-addictive therapies for chronic pan conditions. Voltage-gated sodium (NaV) channel, NaV1.7, is a key player in pain signaling pathway, making it a promising target for novel pain therapeutics. Achieving high subtype selectivity when targeting NaV channels is of primary importance to avoid impairing vital physiological functions mediated by off-target channels. Efforts to selectively target NaV1.7 have been hindered by the difficulties in targeting NaV1.7 over other NaV channel subtypes. Peptidic gating modifier toxins (GMTs), such as Protoxin-II (ProTx2), are promising scaffolds for novel peptide design targeting ion channels with high potency and subtype selectivity. ProTx2 binds to the second and fourth voltage-sensing domains (VSDII and VSDIV) from NaV1.7 with moderate subtype selectivity and can modulate channel activation and inactivation. In this project, we modeled ProTx2 bound to human NaV1.7 VSDIV in an activated state. We used RoseTTAFold Diffusion and Protein MPNN protein design methods to generate protein binders inspired by ProTx2 binding motif with increased predicted binding affinity for human NaV1.7 VSDIV in an activated state. Additionally, we applied these protein design methods to create de novo binders targeting human NaV1.7 VSDIV in an activated state. We anticipate that trapping the VSDIV in an activated conformation will stabilize an inactivated state of the channel, as activation of VSDIV is coupled with channel fast inactivation. Initial electrophysiological screening of our top in silico binders identified promising candidates that inhibited NaV1.7 in the micromolar range. These binders will undergo further testing and optimization against NaV1.7 to create novel molecular tools to study NaV channel activity and effective and safe therapies for chronic pain.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Chronic pain affects about 20% of the US population, but safe treatments are limited. There is an urgent need for effective and non-addictive therapies for chronic pan conditions. Voltage-gated sodium (NaV) channel, NaV1.7, is a key player in pain signaling pathway, making it a promising target for novel pain therapeutics. Achieving high subtype selectivity when targeting NaV channels is of primary importance to avoid impairing vital physiological functions mediated by off-target channels. Efforts to selectively target NaV1.7 have been hindered by the difficulties in targeting NaV1.7 over other NaV channel subtypes. Peptidic gating modifier toxins (GMTs), such as Protoxin-II (ProTx2), are promising scaffolds for novel peptide design targeting ion channels with high potency and subtype selectivity. ProTx2 binds to the second and fourth voltage-sensing domains (VSDII and VSDIV) from NaV1.7 with moderate subtype selectivity and can modulate channel activation and inactivation. In this project, we modeled ProTx2 bound to human NaV1.7 VSDIV in an activated state. We used RoseTTAFold Diffusion and Protein MPNN protein design methods to generate protein binders inspired by ProTx2 binding motif with increased predicted binding affinity for human NaV1.7 VSDIV in an activated state. Additionally, we applied these protein design methods to create de novo binders targeting human NaV1.7 VSDIV in an activated state. We anticipate that trapping the VSDIV in an activated conformation will stabilize an inactivated state of the channel, as activation of VSDIV is coupled with channel fast inactivation. Initial electrophysiological screening of our top in silico binders identified promising candidates that inhibited NaV1.7 in the micromolar range. These binders will undergo further testing and optimization against NaV1.7 to create novel molecular tools to study NaV channel activity and effective and safe therapies for chronic pain.
2023
FROM THE LAB
Susana Vázquez Torres, Philip J Y Leung, Preetham Venkatesh, Isaac D Lutz, Fabian Hink, Huu-Hien Huynh, Jessica Becker, Andy Hsien-Wei Yeh, David Juergens, Nathaniel R Bennett, Andrew N Hoofnagle, Eric Huang, Michael J MacCoss, Marc Expòsit, Gyu Rie Lee, Asim K Bera, Alex Kang, Joshmyn De La Cruz, Paul M Levine, Xinting Li, Mila Lamb, Stacey R Gerben, Analisa Murray, Piper Heine, Elif Nihal Korkmaz, Jeff Nivala, Lance Stewart, Joseph L Watson, Joseph M Rogers, David Baker
De novo design of high-affinity binders of bioactive helical peptides Journal Article
In: Nature, 2023, ISSN: 1476-4687.
@article{pmid38109936,
title = {De novo design of high-affinity binders of bioactive helical peptides},
author = {Susana Vázquez Torres and Philip J Y Leung and Preetham Venkatesh and Isaac D Lutz and Fabian Hink and Huu-Hien Huynh and Jessica Becker and Andy Hsien-Wei Yeh and David Juergens and Nathaniel R Bennett and Andrew N Hoofnagle and Eric Huang and Michael J MacCoss and Marc Expòsit and Gyu Rie Lee and Asim K Bera and Alex Kang and Joshmyn De La Cruz and Paul M Levine and Xinting Li and Mila Lamb and Stacey R Gerben and Analisa Murray and Piper Heine and Elif Nihal Korkmaz and Jeff Nivala and Lance Stewart and Joseph L Watson and Joseph M Rogers and David Baker},
url = {https://www.nature.com/articles/s41586-023-06953-1, Nature [Open Access]},
doi = {10.1038/s41586-023-06953-1},
issn = {1476-4687},
year = {2023},
date = {2023-12-01},
urldate = {2023-12-01},
journal = {Nature},
abstract = {Many peptide hormones form an alpha-helix upon binding their receptors, and sensitive detection methods for them could contribute to better clinical management of disease. De novo protein design can now generate binders with high affinity and specificity to structured proteins. However, the design of interactions between proteins and short peptides with helical propensity is an unmet challenge. Here, we describe parametric generation and deep learning-based methods for designing proteins to address this challenge. We show that by extending RFdiffusion to enable binder design to flexible targets, and to refining input structure models by successive noising and denoising (partial diffusion), picomolar affinity binders can be generated to helical peptide targets both by refining designs generated with other methods, or completely de novo starting from random noise distributions. To our knowledge these are the highest affinity designed binding proteins against any protein or small molecule target generated directly by computation without any experimental optimisation. The RFdiffusion designs enable the enrichment and subsequent detection of parathyroid hormone and glucagon by mass spectrometry, and the construction of bioluminescence-based protein biosensors. The ability to design binders to conformationally variable targets, and to optimise by partial diffusion both natural and designed proteins, should be broadly useful.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Many peptide hormones form an alpha-helix upon binding their receptors, and sensitive detection methods for them could contribute to better clinical management of disease. De novo protein design can now generate binders with high affinity and specificity to structured proteins. However, the design of interactions between proteins and short peptides with helical propensity is an unmet challenge. Here, we describe parametric generation and deep learning-based methods for designing proteins to address this challenge. We show that by extending RFdiffusion to enable binder design to flexible targets, and to refining input structure models by successive noising and denoising (partial diffusion), picomolar affinity binders can be generated to helical peptide targets both by refining designs generated with other methods, or completely de novo starting from random noise distributions. To our knowledge these are the highest affinity designed binding proteins against any protein or small molecule target generated directly by computation without any experimental optimisation. The RFdiffusion designs enable the enrichment and subsequent detection of parathyroid hormone and glucagon by mass spectrometry, and the construction of bioluminescence-based protein biosensors. The ability to design binders to conformationally variable targets, and to optimise by partial diffusion both natural and designed proteins, should be broadly useful.
Edin Muratspahić, Kristine Deibler, Jianming Han, Nataša Tomašević, Kirtikumar B Jadhav, Aina-Leonor Olivé-Marti, Nadine Hochrainer, Roland Hellinger, Johannes Koehbach, Jonathan F Fay, Mohammad Homaidur Rahman, Lamees Hegazy, Timothy W Craven, Balazs R Varga, Gaurav Bhardwaj, Kevin Appourchaux, Susruta Majumdar, Markus Muttenthaler, Parisa Hosseinzadeh, David J Craik, Mariana Spetea, Tao Che, David Baker, Christian W Gruber
Design and structural validation of peptide-drug conjugate ligands of the kappa-opioid receptor Journal Article
In: Nature Communications, 2023.
@article{Muratspahić2023,
title = {Design and structural validation of peptide-drug conjugate ligands of the kappa-opioid receptor},
author = {Edin Muratspahić and Kristine Deibler and Jianming Han and Nataša Tomašević and Kirtikumar B Jadhav and Aina-Leonor Olivé-Marti and Nadine Hochrainer and Roland Hellinger and Johannes Koehbach and Jonathan F Fay and Mohammad Homaidur Rahman and Lamees Hegazy and Timothy W Craven and Balazs R Varga and Gaurav Bhardwaj and Kevin Appourchaux and Susruta Majumdar and Markus Muttenthaler and Parisa Hosseinzadeh and David J Craik and Mariana Spetea and Tao Che and David Baker and Christian W Gruber},
url = {https://www.nature.com/articles/s41467-023-43718-w, Nature Communications [Open Access]},
doi = {10.1038/s41467-023-43718-w},
year = {2023},
date = {2023-12-01},
urldate = {2023-12-01},
journal = {Nature Communications},
abstract = {Despite the increasing number of GPCR structures and recent advances in peptide design, the development of efficient technologies allowing rational design of high-affinity peptide ligands for single GPCRs remains an unmet challenge. Here, we develop a computational approach for designing conjugates of lariat-shaped macrocyclized peptides and a small molecule opioid ligand. We demonstrate its feasibility by discovering chemical scaffolds for the kappa-opioid receptor (KOR) with desired pharmacological activities. The designed De Novo Cyclic Peptide (DNCP)-β-naloxamine (NalA) exhibit in vitro potent mixed KOR agonism/mu-opioid receptor (MOR) antagonism, nanomolar binding affinity, selectivity, and efficacy bias at KOR. Proof-of-concept in vivo efficacy studies demonstrate that DNCP-β-NalA(1) induces a potent KOR-mediated antinociception in male mice. The high-resolution cryo-EM structure (2.6 Å) of the DNCP-β-NalA-KOR-Gi1 complex and molecular dynamics simulations are harnessed to validate the computational design model. This reveals a network of residues in ECL2/3 and TM6/7 controlling the intrinsic efficacy of KOR. In general, our computational de novo platform overcomes extensive lead optimization encountered in ultra-large library docking and virtual small molecule screening campaigns and offers innovation for GPCR ligand discovery. This may drive the development of next-generation therapeutics for medical applications such as pain conditions.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Despite the increasing number of GPCR structures and recent advances in peptide design, the development of efficient technologies allowing rational design of high-affinity peptide ligands for single GPCRs remains an unmet challenge. Here, we develop a computational approach for designing conjugates of lariat-shaped macrocyclized peptides and a small molecule opioid ligand. We demonstrate its feasibility by discovering chemical scaffolds for the kappa-opioid receptor (KOR) with desired pharmacological activities. The designed De Novo Cyclic Peptide (DNCP)-β-naloxamine (NalA) exhibit in vitro potent mixed KOR agonism/mu-opioid receptor (MOR) antagonism, nanomolar binding affinity, selectivity, and efficacy bias at KOR. Proof-of-concept in vivo efficacy studies demonstrate that DNCP-β-NalA(1) induces a potent KOR-mediated antinociception in male mice. The high-resolution cryo-EM structure (2.6 Å) of the DNCP-β-NalA-KOR-Gi1 complex and molecular dynamics simulations are harnessed to validate the computational design model. This reveals a network of residues in ECL2/3 and TM6/7 controlling the intrinsic efficacy of KOR. In general, our computational de novo platform overcomes extensive lead optimization encountered in ultra-large library docking and virtual small molecule screening campaigns and offers innovation for GPCR ligand discovery. This may drive the development of next-generation therapeutics for medical applications such as pain conditions.
Florian Praetorius, Philip J. Y. Leung, Maxx H. Tessmer, Adam Broerman, Cullen Demakis, Acacia F. Dishman, Arvind Pillai, Abbas Idris, David Juergens, Justas Dauparas, Xinting Li, Paul M. Levine, Mila Lamb, Ryanne K. Ballard, Stacey R. Gerben, Hannah Nguyen, Alex Kang, Banumathi Sankaran, Asim K. Bera, Brian F. Volkman, Jeff Nivala, Stefan Stoll, David Baker
Design of stimulus-responsive two-state hinge proteins Journal Article
In: Science, 2023.
@article{Praetorius2023,
title = {Design of stimulus-responsive two-state hinge proteins},
author = {Florian Praetorius and Philip J. Y. Leung and Maxx H. Tessmer and Adam Broerman and Cullen Demakis and Acacia F. Dishman and Arvind Pillai and Abbas Idris and David Juergens and Justas Dauparas and Xinting Li and Paul M. Levine and Mila Lamb and Ryanne K. Ballard and Stacey R. Gerben and Hannah Nguyen and Alex Kang and Banumathi Sankaran and Asim K. Bera and Brian F. Volkman and Jeff Nivala and Stefan Stoll and David Baker},
url = {https://www.science.org/stoken/author-tokens/ST-1381/full, Science (Free Access)},
doi = {10.1126/science.adg7731},
year = {2023},
date = {2023-08-17},
urldate = {2023-08-17},
journal = {Science},
abstract = {In nature, proteins that switch between two conformations in response to environmental stimuli structurally transduce biochemical information in a manner analogous to how transistors control information flow in computing devices. Designing proteins with two distinct but fully structured conformations is a challenge for protein design as it requires sculpting an energy landscape with two distinct minima. Here we describe the design of “hinge” proteins that populate one designed state in the absence of ligand and a second designed state in the presence of ligand. X-ray crystallography, electron microscopy, double electron-electron resonance spectroscopy, and binding measurements demonstrate that despite the significant structural differences the two states are designed with atomic level accuracy and that the conformational and binding equilibria are closely coupled. Natural proteins often adopt multiple conformational states, thereby changing their activity or binding partners in response to another protein, small molecule, or other stimulus. It has been difficult to engineer such conformational switching between two folded states in human-designed proteins. Praetorius et al. developed a hinge-like protein by simultaneously considering both desired states in the design process. The successful designs exhibited a large shift in conformation upon binding to a target peptide helix, which could be tailored for specificity. The authors characterized the protein structures, binding kinetics, and conformational equilibrium of the designs. This work provides the groundwork for generating protein switches that respond to biological triggers and can produce conformational changes that modulate protein assemblies. —Michael A. Funk A two-state design of protein switches that couple effector binding to a conformational change is discussed.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In nature, proteins that switch between two conformations in response to environmental stimuli structurally transduce biochemical information in a manner analogous to how transistors control information flow in computing devices. Designing proteins with two distinct but fully structured conformations is a challenge for protein design as it requires sculpting an energy landscape with two distinct minima. Here we describe the design of “hinge” proteins that populate one designed state in the absence of ligand and a second designed state in the presence of ligand. X-ray crystallography, electron microscopy, double electron-electron resonance spectroscopy, and binding measurements demonstrate that despite the significant structural differences the two states are designed with atomic level accuracy and that the conformational and binding equilibria are closely coupled. Natural proteins often adopt multiple conformational states, thereby changing their activity or binding partners in response to another protein, small molecule, or other stimulus. It has been difficult to engineer such conformational switching between two folded states in human-designed proteins. Praetorius et al. developed a hinge-like protein by simultaneously considering both desired states in the design process. The successful designs exhibited a large shift in conformation upon binding to a target peptide helix, which could be tailored for specificity. The authors characterized the protein structures, binding kinetics, and conformational equilibrium of the designs. This work provides the groundwork for generating protein switches that respond to biological triggers and can produce conformational changes that modulate protein assemblies. —Michael A. Funk A two-state design of protein switches that couple effector binding to a conformational change is discussed.
Wu, Kejia and Bai, Hua and Chang, Ya-Ting and Redler, Rachel and McNally, Kerrie E. and Sheffler, William and Brunette, T. J. and Hicks, Derrick R. and Morgan, Tomos E. and Stevens, Tim J. and Broerman, Adam and Goreshnik, Inna and DeWitt, Michelle and Chow, Cameron M. and Shen, Yihang and Stewart, Lance and Derivery, Emmanuel and Silva, Daniel Adriano and Bhabha, Gira and Ekiert, Damian C. and Baker, David
De novo design of modular peptide-binding proteins by superhelical matching Journal Article
In: Nature, 2023.
@article{Wu2023,
title = {De novo design of modular peptide-binding proteins by superhelical matching},
author = {Wu, Kejia
and Bai, Hua
and Chang, Ya-Ting
and Redler, Rachel
and McNally, Kerrie E.
and Sheffler, William
and Brunette, T. J.
and Hicks, Derrick R.
and Morgan, Tomos E.
and Stevens, Tim J.
and Broerman, Adam
and Goreshnik, Inna
and DeWitt, Michelle
and Chow, Cameron M.
and Shen, Yihang
and Stewart, Lance
and Derivery, Emmanuel
and Silva, Daniel Adriano
and Bhabha, Gira
and Ekiert, Damian C.
and Baker, David},
url = {https://www.nature.com/articles/s41586-023-05909-9, Nature (Open-access)},
doi = {10.1038/s41586-023-05909-9},
year = {2023},
date = {2023-04-05},
urldate = {2023-04-05},
journal = {Nature},
abstract = {General approaches for designing sequence-specific peptide-binding proteins would have wide utility in proteomics and synthetic biology. However, designing peptide-binding proteins is challenging, as most peptides do not have defined structures in isolation, and hydrogen bonds must be made to the buried polar groups in the peptide backbone1–3. Here, inspired by natural and re-engineered protein–peptide systems4–11, we set out to design proteins made out of repeating units that bind peptides with repeating sequences, with a one-to-one correspondence between the repeat units of the protein and those of the peptide. We use geometric hashing to identify protein backbones and peptide-docking arrangements that are compatible with bidentate hydrogen bonds between the side chains of the protein and the peptide backbone12. The remainder of the protein sequence is then optimized for folding and peptide binding. We design repeat proteins to bind to six different tripeptide-repeat sequences in polyproline II conformations. The proteins are hyperstable and bind to four to six tandem repeats of their tripeptide targets with nanomolar to picomolar affinities in vitro and in living cells. Crystal structures reveal repeating interactions between protein and peptide interactions as designed, including ladders of hydrogen bonds from protein side chains to peptide backbones. By redesigning the binding interfaces of individual repeat units, specificity can be achieved for non-repeating peptide sequences and for disordered regions of native proteins.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
General approaches for designing sequence-specific peptide-binding proteins would have wide utility in proteomics and synthetic biology. However, designing peptide-binding proteins is challenging, as most peptides do not have defined structures in isolation, and hydrogen bonds must be made to the buried polar groups in the peptide backbone1–3. Here, inspired by natural and re-engineered protein–peptide systems4–11, we set out to design proteins made out of repeating units that bind peptides with repeating sequences, with a one-to-one correspondence between the repeat units of the protein and those of the peptide. We use geometric hashing to identify protein backbones and peptide-docking arrangements that are compatible with bidentate hydrogen bonds between the side chains of the protein and the peptide backbone12. The remainder of the protein sequence is then optimized for folding and peptide binding. We design repeat proteins to bind to six different tripeptide-repeat sequences in polyproline II conformations. The proteins are hyperstable and bind to four to six tandem repeats of their tripeptide targets with nanomolar to picomolar affinities in vitro and in living cells. Crystal structures reveal repeating interactions between protein and peptide interactions as designed, including ladders of hydrogen bonds from protein side chains to peptide backbones. By redesigning the binding interfaces of individual repeat units, specificity can be achieved for non-repeating peptide sequences and for disordered regions of native proteins.
Amir Motmaen, Justas Dauparas, Minkyung Baek, Mohamad H. Abedi, David Baker, Philip Bradley
Peptide-binding specificity prediction using fine-tuned protein structure prediction networks Journal Article
In: Proceedings of the National Academy of Sciences, 2023.
@article{nokey,
title = {Peptide-binding specificity prediction using fine-tuned protein structure prediction networks},
author = {Amir Motmaen, Justas Dauparas, Minkyung Baek, Mohamad H. Abedi, David Baker, Philip Bradley},
url = {https://www.pnas.org/doi/10.1073/pnas.2216697120, PNAS (Open Access)},
doi = {10.1073/pnas.2216697120},
year = {2023},
date = {2023-02-21},
urldate = {2023-02-21},
journal = {Proceedings of the National Academy of Sciences},
abstract = {Peptide-binding proteins play key roles in biology, and predicting their binding specificity is a long-standing challenge. While considerable protein structural information is available, the most successful current methods use sequence information alone, in part because it has been a challenge to model the subtle structural changes accompanying sequence substitutions. Protein structure prediction networks such as AlphaFold model sequence-structure relationships very accurately, and we reasoned that if it were possible to specifically train such networks on binding data, more generalizable models could be created. We show that placing a classifier on top of the AlphaFold network and fine-tuning the combined network parameters for both classification and structure prediction accuracy leads to a model with strong generalizable performance on a wide range of Class I and Class II peptide-MHC interactions that approaches the overall performance of the state-of-the-art NetMHCpan sequence-based method. The peptide-MHC optimized model shows excellent performance in distinguishing binding and non-binding peptides to SH3 and PDZ domains. This ability to generalize well beyond the training set far exceeds that of sequence-only models and should be particularly powerful for systems where less experimental data are available.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Peptide-binding proteins play key roles in biology, and predicting their binding specificity is a long-standing challenge. While considerable protein structural information is available, the most successful current methods use sequence information alone, in part because it has been a challenge to model the subtle structural changes accompanying sequence substitutions. Protein structure prediction networks such as AlphaFold model sequence-structure relationships very accurately, and we reasoned that if it were possible to specifically train such networks on binding data, more generalizable models could be created. We show that placing a classifier on top of the AlphaFold network and fine-tuning the combined network parameters for both classification and structure prediction accuracy leads to a model with strong generalizable performance on a wide range of Class I and Class II peptide-MHC interactions that approaches the overall performance of the state-of-the-art NetMHCpan sequence-based method. The peptide-MHC optimized model shows excellent performance in distinguishing binding and non-binding peptides to SH3 and PDZ domains. This ability to generalize well beyond the training set far exceeds that of sequence-only models and should be particularly powerful for systems where less experimental data are available.
COLLABORATOR LED
Enrico Rennella, Danny D. Sahtoe, David Baker,, Lewis E. Kay
Exploiting conformational dynamics to modulate the function of designed proteins Journal Article
In: Proceedings of the National Academy of Sciences, 2023.
@article{nokey,
title = {Exploiting conformational dynamics to modulate the function of designed proteins},
author = {Enrico Rennella, Danny D. Sahtoe, David Baker, and Lewis E. Kay
},
url = {https://www.pnas.org/doi/10.1073/pnas.2303149120, PNAS},
doi = {10.1073/pnas.2303149120},
year = {2023},
date = {2023-04-24},
journal = {Proceedings of the National Academy of Sciences},
abstract = {With the recent success in calculating protein structures from amino acid sequences using artificial intelligence-based algorithms, an important next step is to decipher how dynamics is encoded by the primary protein sequence so as to better predict function. Such dynamics information is critical for protein design, where strategies could then focus not only on sequences that fold into particular structures that perform a given task, but would also include low-lying excited protein states that could influence the function of the designed protein. Herein, we illustrate the importance of dynamics in modulating the function of C34, a designed α/β protein that captures β-strands of target ligands and is a member of a family of proteins designed to sequester β-strands and β hairpins of aggregation-prone molecules that lead to a variety of pathologies. Using a strategy to “see” regions of apo C34 that are invisible to NMR spectroscopy as a result of pervasive conformational exchange, as well as a mutagenesis approach whereby C34 molecules are stabilized into a single conformer, we determine the structures of the predominant conformations that are sampled by C34 and show that these attenuate the affinity for cognate peptide. Subsequently, the observed motion is exploited to develop an allosterically regulated peptide binder whose binding affinity can be controlled through the addition of a second molecule. Our study emphasizes the unique role that NMR can play in directing the design process and in the construction of new molecules with more complex functionality.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
With the recent success in calculating protein structures from amino acid sequences using artificial intelligence-based algorithms, an important next step is to decipher how dynamics is encoded by the primary protein sequence so as to better predict function. Such dynamics information is critical for protein design, where strategies could then focus not only on sequences that fold into particular structures that perform a given task, but would also include low-lying excited protein states that could influence the function of the designed protein. Herein, we illustrate the importance of dynamics in modulating the function of C34, a designed α/β protein that captures β-strands of target ligands and is a member of a family of proteins designed to sequester β-strands and β hairpins of aggregation-prone molecules that lead to a variety of pathologies. Using a strategy to “see” regions of apo C34 that are invisible to NMR spectroscopy as a result of pervasive conformational exchange, as well as a mutagenesis approach whereby C34 molecules are stabilized into a single conformer, we determine the structures of the predominant conformations that are sampled by C34 and show that these attenuate the affinity for cognate peptide. Subsequently, the observed motion is exploited to develop an allosterically regulated peptide binder whose binding affinity can be controlled through the addition of a second molecule. Our study emphasizes the unique role that NMR can play in directing the design process and in the construction of new molecules with more complex functionality.
Watson, Paris R. and Gupta, Suchetana and Hosseinzadeh, Parisa and Brown, Benjamin P. and Baker, David and Christianson, David W.
Macrocyclic Octapeptide Binding and Inferences on Protein Substrate Binding to Histone Deacetylase 6 Journal Article
In: ACS Chemical Biology, 2023.
@article{Watson0000,
title = {Macrocyclic Octapeptide Binding and Inferences on Protein Substrate Binding to Histone Deacetylase 6},
author = {Watson, Paris R.
and Gupta, Suchetana
and Hosseinzadeh, Parisa
and Brown, Benjamin P.
and Baker, David
and Christianson, David W.},
url = {https://pubs.acs.org/doi/full/10.1021/acschembio.3c00113, ACS Chem. Biol.
https://www.bakerlab.org/wp-content/uploads/2023/04/acschembio.3c00113.pdf, PDF},
doi = {10.1021/acschembio.3c00113},
year = {2023},
date = {2023-04-07},
urldate = {2023-04-07},
journal = {ACS Chemical Biology},
abstract = {Histone deacetylases (HDACs) are essential for the regulation of myriad biological processes, and their aberrant function is implicated in cancer, neurodegeneration, and other diseases. The cytosolic isozyme HDAC6 is unique among the greater family of deacetylases in that it contains two catalytic domains, CD1 and CD2. HDAC6 CD2 is responsible for tubulin deacetylase and tau deacetylase activities, inhibition of which is a key goal as new therapeutic approaches are explored. Of particular interest as HDAC inhibitors are naturally occurring cyclic tetrapeptides such as Trapoxin A or HC Toxin, or the cyclic depsipeptides Largazole and Romidepsin. Even more intriguing are larger, computationally designed macrocyclic peptide inhibitors. Here, we report the 2.0 Å resolution crystal structure of HDAC6 CD2 complexed with macrocyclic octapeptide 1. Comparison with the previously reported structure of the complex with macrocyclic octapeptide 2 reveals that a potent thiolate–zinc interaction made by the unnatural amino acid (S)-2-amino-7-sulfanylheptanoic acid contributes to nanomolar inhibitory potency for each inhibitor. Apart from this zinc-binding residue, octapeptides adopt strikingly different overall conformations and make few direct hydrogen bonds with the protein. Intermolecular interactions are dominated by water-mediated hydrogen bonds; in essence, water molecules appear to cushion the enzyme–octapeptide interface. In view of the broad specificity observed for protein substrates of HDAC6 CD2, we suggest that the binding of macrocyclic octapeptides may mimic certain features of the binding of macromolecular protein substrates.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Histone deacetylases (HDACs) are essential for the regulation of myriad biological processes, and their aberrant function is implicated in cancer, neurodegeneration, and other diseases. The cytosolic isozyme HDAC6 is unique among the greater family of deacetylases in that it contains two catalytic domains, CD1 and CD2. HDAC6 CD2 is responsible for tubulin deacetylase and tau deacetylase activities, inhibition of which is a key goal as new therapeutic approaches are explored. Of particular interest as HDAC inhibitors are naturally occurring cyclic tetrapeptides such as Trapoxin A or HC Toxin, or the cyclic depsipeptides Largazole and Romidepsin. Even more intriguing are larger, computationally designed macrocyclic peptide inhibitors. Here, we report the 2.0 Å resolution crystal structure of HDAC6 CD2 complexed with macrocyclic octapeptide 1. Comparison with the previously reported structure of the complex with macrocyclic octapeptide 2 reveals that a potent thiolate–zinc interaction made by the unnatural amino acid (S)-2-amino-7-sulfanylheptanoic acid contributes to nanomolar inhibitory potency for each inhibitor. Apart from this zinc-binding residue, octapeptides adopt strikingly different overall conformations and make few direct hydrogen bonds with the protein. Intermolecular interactions are dominated by water-mediated hydrogen bonds; in essence, water molecules appear to cushion the enzyme–octapeptide interface. In view of the broad specificity observed for protein substrates of HDAC6 CD2, we suggest that the binding of macrocyclic octapeptides may mimic certain features of the binding of macromolecular protein substrates.
Florian Praetorius, Philip J. Y. Leung, Maxx H. Tessmer, Adam Broerman, Cullen Demakis, Acacia F. Dishman, Arvind Pillai, Abbas Idris, David Juergens, Justas Dauparas, Xinting Li, Paul M. Levine, Mila Lamb, Ryanne K. Ballard, Stacey R. Gerben, Hannah Nguyen, Alex Kang, Banumathi Sankaran, Asim K. Bera, Brian F. Volkman, Jeff Nivala, Stefan Stoll, David Baker
Design of stimulus-responsive two-state hinge proteins Journal Article
In: Science, vol. 381, no. 6659, pp. 754-760, 2023.
@article{doi:10.1126/science.adg7731b,
title = {Design of stimulus-responsive two-state hinge proteins},
author = {Florian Praetorius and Philip J. Y. Leung and Maxx H. Tessmer and Adam Broerman and Cullen Demakis and Acacia F. Dishman and Arvind Pillai and Abbas Idris and David Juergens and Justas Dauparas and Xinting Li and Paul M. Levine and Mila Lamb and Ryanne K. Ballard and Stacey R. Gerben and Hannah Nguyen and Alex Kang and Banumathi Sankaran and Asim K. Bera and Brian F. Volkman and Jeff Nivala and Stefan Stoll and David Baker},
url = {https://www.science.org/doi/abs/10.1126/science.adg7731},
doi = {10.1126/science.adg7731},
year = {2023},
date = {2023-01-01},
journal = {Science},
volume = {381},
number = {6659},
pages = {754-760},
abstract = {In nature, proteins that switch between two conformations in response to environmental stimuli structurally transduce biochemical information in a manner analogous to how transistors control information flow in computing devices. Designing proteins with two distinct but fully structured conformations is a challenge for protein design as it requires sculpting an energy landscape with two distinct minima. Here we describe the design of “hinge” proteins that populate one designed state in the absence of ligand and a second designed state in the presence of ligand. X-ray crystallography, electron microscopy, double electron-electron resonance spectroscopy, and binding measurements demonstrate that despite the significant structural differences the two states are designed with atomic level accuracy and that the conformational and binding equilibria are closely coupled. Natural proteins often adopt multiple conformational states, thereby changing their activity or binding partners in response to another protein, small molecule, or other stimulus. It has been difficult to engineer such conformational switching between two folded states in human-designed proteins. Praetorius et al. developed a hinge-like protein by simultaneously considering both desired states in the design process. The successful designs exhibited a large shift in conformation upon binding to a target peptide helix, which could be tailored for specificity. The authors characterized the protein structures, binding kinetics, and conformational equilibrium of the designs. This work provides the groundwork for generating protein switches that respond to biological triggers and can produce conformational changes that modulate protein assemblies. —Michael A. Funk A two-state design of protein switches that couple effector binding to a conformational change is discussed.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In nature, proteins that switch between two conformations in response to environmental stimuli structurally transduce biochemical information in a manner analogous to how transistors control information flow in computing devices. Designing proteins with two distinct but fully structured conformations is a challenge for protein design as it requires sculpting an energy landscape with two distinct minima. Here we describe the design of “hinge” proteins that populate one designed state in the absence of ligand and a second designed state in the presence of ligand. X-ray crystallography, electron microscopy, double electron-electron resonance spectroscopy, and binding measurements demonstrate that despite the significant structural differences the two states are designed with atomic level accuracy and that the conformational and binding equilibria are closely coupled. Natural proteins often adopt multiple conformational states, thereby changing their activity or binding partners in response to another protein, small molecule, or other stimulus. It has been difficult to engineer such conformational switching between two folded states in human-designed proteins. Praetorius et al. developed a hinge-like protein by simultaneously considering both desired states in the design process. The successful designs exhibited a large shift in conformation upon binding to a target peptide helix, which could be tailored for specificity. The authors characterized the protein structures, binding kinetics, and conformational equilibrium of the designs. This work provides the groundwork for generating protein switches that respond to biological triggers and can produce conformational changes that modulate protein assemblies. —Michael A. Funk A two-state design of protein switches that couple effector binding to a conformational change is discussed.
2022
FROM THE LAB
Said, Meerit Y., Kang, Christine S., Wang, Shunzhi, Sheffler, William, Salveson, Patrick J., Bera, Asim K., Kang, Alex, Nguyen, Hannah, Ballard, Ryanne, Li, Xinting, Bai, Hua, Stewart, Lance, Levine, Paul, Baker, David
Exploration of Structured Symmetric Cyclic Peptides as Ligands for Metal-Organic Frameworks Journal Article
In: Chemistry of Materials, 2022.
@article{Said2022,
title = {Exploration of Structured Symmetric Cyclic Peptides as Ligands for Metal-Organic Frameworks},
author = {Said, Meerit Y. and Kang, Christine S. and Wang, Shunzhi and Sheffler, William and Salveson, Patrick J. and Bera, Asim K. and Kang, Alex and Nguyen, Hannah and Ballard, Ryanne and Li, Xinting and Bai, Hua and Stewart, Lance and Levine, Paul and Baker, David},
url = {https://pubs.acs.org/doi/10.1021/acs.chemmater.2c02597, Chem. Mater.
https://www.bakerlab.org/wp-content/uploads/2022/10/Said_etal_ChemMater2022_CyclicPeptideMOFs.pdf, PDF},
doi = {/10.1021/acs.chemmater.2c02597},
year = {2022},
date = {2022-10-25},
urldate = {2022-10-25},
journal = {Chemistry of Materials},
abstract = {Despite remarkable advances in the assembly of highly structured coordination polymers and metal–organic frameworks, the rational design of such materials using more conformationally flexible organic ligands such as peptides remains challenging. In an effort to make the design of such materials fully programmable, we first developed a computational design method for generating metal-mediated 3D frameworks using rigid and symmetric peptide macrocycles with metal-coordinating sidechains. We solved the structures of six crystalline networks involving conformationally constrained 6 to 12 residue cyclic peptides with C2, C3, and S2 internal symmetry and three different types of metals (Zn2+, Co2+, or Cu2+) by single-crystal X-ray diffraction, which reveals how the peptide sequences, backbone symmetries, and metal coordination preferences drive the assembly of the resulting structures. In contrast to smaller ligands, these peptides associate through peptide–peptide interactions without full coordination of the metals, contrary to one of the assumptions underlying our computational design method. The cyclic peptides are the largest peptidic ligands reported to form crystalline coordination polymers with transition metals to date, and while more work is required to develop methods for fully programming their crystal structures, the combination of high chemical diversity with synthetic accessibility makes them attractive building blocks for engineering a broader set of new crystalline materials for use in applications such as sensing, asymmetric catalysis, and chiral separation.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Despite remarkable advances in the assembly of highly structured coordination polymers and metal–organic frameworks, the rational design of such materials using more conformationally flexible organic ligands such as peptides remains challenging. In an effort to make the design of such materials fully programmable, we first developed a computational design method for generating metal-mediated 3D frameworks using rigid and symmetric peptide macrocycles with metal-coordinating sidechains. We solved the structures of six crystalline networks involving conformationally constrained 6 to 12 residue cyclic peptides with C2, C3, and S2 internal symmetry and three different types of metals (Zn2+, Co2+, or Cu2+) by single-crystal X-ray diffraction, which reveals how the peptide sequences, backbone symmetries, and metal coordination preferences drive the assembly of the resulting structures. In contrast to smaller ligands, these peptides associate through peptide–peptide interactions without full coordination of the metals, contrary to one of the assumptions underlying our computational design method. The cyclic peptides are the largest peptidic ligands reported to form crystalline coordination polymers with transition metals to date, and while more work is required to develop methods for fully programming their crystal structures, the combination of high chemical diversity with synthetic accessibility makes them attractive building blocks for engineering a broader set of new crystalline materials for use in applications such as sensing, asymmetric catalysis, and chiral separation.
Gaurav Bhardwaj, Jacob O’Connor, Stephen Rettie, Yen-Hua Huang, Theresa A. Ramelot, Vikram Khipple Mulligan, Gizem Gokce Alpkilic, Jonathan Palmer, Asim K. Bera, Matthew J. Bick, Maddalena Di Piazza, Xinting Li, Parisa Hosseinzadeh, Timothy W. Craven, Roberto Tejero, Anna Lauko, Ryan Choi, Calina Glynn, Linlin Dong, Robert Griffin, Wesley C. van Voorhis, Jose Rodriguez, Lance Stewart, Gaetano T. Montelione, David Craik, David Baker
Accurate de novo design of membrane-traversing macrocycles Journal Article
In: Cell, 2022.
@article{Bhardwaj2022,
title = {Accurate de novo design of membrane-traversing macrocycles},
author = {Gaurav Bhardwaj and Jacob O’Connor and Stephen Rettie and Yen-Hua Huang and Theresa A. Ramelot and Vikram Khipple Mulligan and Gizem Gokce Alpkilic and Jonathan Palmer and Asim K. Bera and Matthew J. Bick and Maddalena {Di Piazza} and Xinting Li and Parisa Hosseinzadeh and Timothy W. Craven and Roberto Tejero and Anna Lauko and Ryan Choi and Calina Glynn and Linlin Dong and Robert Griffin and Wesley C. {van Voorhis} and Jose Rodriguez and Lance Stewart and Gaetano T. Montelione and David Craik and David Baker},
url = {https://www.sciencedirect.com/science/article/pii/S0092867422009229?via%3Dihub, Cell
https://www.bakerlab.org/wp-content/uploads/2022/08/1-s2.0-S0092867422009229-main.pdf, PDF},
doi = {10.1016/j.cell.2022.07.019},
year = {2022},
date = {2022-08-29},
urldate = {2022-08-29},
journal = {Cell},
abstract = {We use computational design coupled with experimental characterization to systematically investigate the design principles for macrocycle membrane permeability and oral bioavailability. We designed 184 6–12 residue macrocycles with a wide range of predicted structures containing noncanonical backbone modifications and experimentally determined structures of 35; 29 are very close to the computational models. With such control, we show that membrane permeability can be systematically achieved by ensuring all amide (NH) groups are engaged in internal hydrogen bonding interactions. 84 designs over the 6–12 residue size range cross membranes with an apparent permeability greater than 1 × 10−6 cm/s. Designs with exposed NH groups can be made membrane permeable through the design of an alternative isoenergetic fully hydrogen-bonded state favored in the lipid membrane. The ability to robustly design membrane-permeable and orally bioavailable peptides with high structural accuracy should contribute to the next generation of designed macrocycle therapeutics.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We use computational design coupled with experimental characterization to systematically investigate the design principles for macrocycle membrane permeability and oral bioavailability. We designed 184 6–12 residue macrocycles with a wide range of predicted structures containing noncanonical backbone modifications and experimentally determined structures of 35; 29 are very close to the computational models. With such control, we show that membrane permeability can be systematically achieved by ensuring all amide (NH) groups are engaged in internal hydrogen bonding interactions. 84 designs over the 6–12 residue size range cross membranes with an apparent permeability greater than 1 × 10−6 cm/s. Designs with exposed NH groups can be made membrane permeable through the design of an alternative isoenergetic fully hydrogen-bonded state favored in the lipid membrane. The ability to robustly design membrane-permeable and orally bioavailable peptides with high structural accuracy should contribute to the next generation of designed macrocycle therapeutics.
Levine, Paul M., Craven, Timothy W., Li, Xinting, Balana, Aaron T., Bird, Gregory H., Godes, Marina, Salveson, Patrick J., Erickson, Patrick W., Lamb, Mila, Ahlrichs, Maggie, Murphy, Michael, Ogohara, Cassandra, Said, Meerit Y., Walensky, Loren D., Pratt, Matthew R., Baker, David
Generation of Potent and Stable GLP-1 Analogues Via “Serine Ligation” Journal Article
In: ACS Chemical Biology, 2022.
@article{nokey,
title = {Generation of Potent and Stable GLP-1 Analogues Via “Serine Ligation”},
author = {Levine, Paul M. and Craven, Timothy W. and Li, Xinting and Balana, Aaron T. and Bird, Gregory H. and Godes, Marina and Salveson, Patrick J. and Erickson, Patrick W. and Lamb, Mila and Ahlrichs, Maggie and Murphy, Michael and Ogohara, Cassandra and Said, Meerit Y. and Walensky, Loren D. and Pratt, Matthew R. and Baker, David},
url = {https://pubs.acs.org/doi/abs/10.1021/acschembio.2c00075, ACS Chemical Biology
https://www.bakerlab.org/wp-content/uploads/2022/03/Levine_etal_ACSChemBio2022_GLP-1_ananlogues_by_serine_ligation.pdf, Download PDF},
doi = {10.1021/acschembio.2c00075},
year = {2022},
date = {2022-03-23},
journal = {ACS Chemical Biology},
abstract = {Peptide and protein bioconjugation technologies have revolutionized our ability to site-specifically or chemoselectively install a variety of functional groups for applications in chemical biology and medicine, including the enhancement of bioavailability. Here, we introduce a site-specific bioconjugation strategy inspired by chemical ligation at serine that relies on a noncanonical amino acid containing a 1-amino-2-hydroxy functional group and a salicylaldehyde ester. More specifically, we harness this technology to generate analogues of glucagon-like peptide-1 that resemble Semaglutide, a long-lasting blockbuster drug currently used in the clinic to regulate glucose levels in the blood. We identify peptides that are more potent than unmodified peptide and equipotent to Semaglutide in a cell-based activation assay, improve the stability in human serum, and increase glucose disposal efficiency in vivo. This approach demonstrates the potential of “serine ligation” for various applications in chemical biology, with a particular focus on generating stabilized peptide therapeutics.
},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Peptide and protein bioconjugation technologies have revolutionized our ability to site-specifically or chemoselectively install a variety of functional groups for applications in chemical biology and medicine, including the enhancement of bioavailability. Here, we introduce a site-specific bioconjugation strategy inspired by chemical ligation at serine that relies on a noncanonical amino acid containing a 1-amino-2-hydroxy functional group and a salicylaldehyde ester. More specifically, we harness this technology to generate analogues of glucagon-like peptide-1 that resemble Semaglutide, a long-lasting blockbuster drug currently used in the clinic to regulate glucose levels in the blood. We identify peptides that are more potent than unmodified peptide and equipotent to Semaglutide in a cell-based activation assay, improve the stability in human serum, and increase glucose disposal efficiency in vivo. This approach demonstrates the potential of “serine ligation” for various applications in chemical biology, with a particular focus on generating stabilized peptide therapeutics.
COLLABORATOR LED
Macé, Kévin and Vadakkepat, Abhinav K. and Redzej, Adam and Lukoyanova, Natalya and Oomen, Clasien and Braun, Nathalie and Ukleja, Marta and Lu, Fang and Costa, Tiago R. D. and Orlova, Elena V. and Baker, David and Cong, Qian and Waksman, Gabriel
Cryo-EM structure of a type IV secretion system Journal Article
In: Nature, 2022.
@article{Macé2022,
title = {Cryo-EM structure of a type IV secretion system},
author = {Macé, Kévin
and Vadakkepat, Abhinav K.
and Redzej, Adam
and Lukoyanova, Natalya
and Oomen, Clasien
and Braun, Nathalie
and Ukleja, Marta
and Lu, Fang
and Costa, Tiago R. D.
and Orlova, Elena V.
and Baker, David
and Cong, Qian
and Waksman, Gabriel},
url = {https://www.nature.com/articles/s41586-022-04859-y, Nature
https://www.bakerlab.org/wp-content/uploads/2022/08/Mace2022s41586-022-04859-y.pdf, PDF},
doi = {10.1038/s41586-022-04859-y},
year = {2022},
date = {2022-07-01},
urldate = {2022-07-01},
journal = {Nature},
abstract = {Bacterial conjugation is the fundamental process of unidirectional transfer of DNAs, often plasmid DNAs, from a donor cell to a recipient cell1. It is the primary means by which antibiotic resistance genes spread among bacterial populations2,3. In Gram-negative bacteria, conjugation is mediated by a large transport apparatus—the conjugative type IV secretion system (T4SS)—produced by the donor cell and embedded in both its outer and inner membranes. The T4SS also elaborates a long extracellular filament—the conjugative pilus—that is essential for DNA transfer4,5. Here we present a high-resolution cryo-electron microscopy (cryo-EM) structure of a 2.8 megadalton T4SS complex composed of 92 polypeptides representing 8 of the 10 essential T4SS components involved in pilus biogenesis. We added the two remaining components to the structural model using co-evolution analysis of protein interfaces, to enable the reconstitution of the entire system including the pilus. This structure describes the exceptionally large protein–protein interaction network required to assemble the many components that constitute a T4SS and provides insights on the unique mechanism by which they elaborate pili.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Bacterial conjugation is the fundamental process of unidirectional transfer of DNAs, often plasmid DNAs, from a donor cell to a recipient cell1. It is the primary means by which antibiotic resistance genes spread among bacterial populations2,3. In Gram-negative bacteria, conjugation is mediated by a large transport apparatus—the conjugative type IV secretion system (T4SS)—produced by the donor cell and embedded in both its outer and inner membranes. The T4SS also elaborates a long extracellular filament—the conjugative pilus—that is essential for DNA transfer4,5. Here we present a high-resolution cryo-electron microscopy (cryo-EM) structure of a 2.8 megadalton T4SS complex composed of 92 polypeptides representing 8 of the 10 essential T4SS components involved in pilus biogenesis. We added the two remaining components to the structural model using co-evolution analysis of protein interfaces, to enable the reconstitution of the entire system including the pilus. This structure describes the exceptionally large protein–protein interaction network required to assemble the many components that constitute a T4SS and provides insights on the unique mechanism by which they elaborate pili.
Yao, Sicong, Moyer, Adam, Zheng, Yiwu, Shen, Yang, Meng, Xiaoting, Yuan, Chong, Zhao, Yibing, Yao, Hongwei, Baker, David, Wu, Chuanliu
De novo design and directed folding of disulfide-bridged peptide heterodimers Journal Article
In: Nature Communications, 2022.
@article{Yao2022,
title = {De novo design and directed folding of disulfide-bridged peptide heterodimers},
author = {Yao, Sicong and Moyer, Adam and Zheng, Yiwu and Shen, Yang and Meng, Xiaoting and Yuan, Chong and Zhao, Yibing and Yao, Hongwei and Baker, David and Wu, Chuanliu},
url = {https://www.nature.com/articles/s41467-022-29210-x, Nature Communications
https://www.bakerlab.org/wp-content/uploads/2022/03/Yao_etal_NatComms2022_Design_of_directed_folding_of_disulfile_bridged_peptide_heterodimers.pdf, Download PDF},
year = {2022},
date = {2022-03-22},
urldate = {2022-03-22},
journal = {Nature Communications},
abstract = {Peptide heterodimers are prevalent in nature, which are not only functional macromolecules but molecular tools for chemical and synthetic biology. Computational methods have also been developed to design heterodimers of advanced functions. However, these peptide heterodimers are usually formed through noncovalent interactions, which are prone to dissociate and subject to concentration-dependent nonspecific aggregation. Heterodimers crosslinked with interchain disulfide bonds are more stable, but it represents a formidable challenge for both the computational design of heterodimers and the manipulation of disulfide pairing for heterodimer synthesis and applications. Here, we report the design, synthesis and application of interchain disulfide-bridged peptide heterodimers with mutual orthogonality by combining computational de novo designs with a directed disulfide pairing strategy. These heterodimers can be used as not only scaffolds for generating functional molecules but chemical tools or building blocks for protein labeling and construction of crosslinking hybrids. This study thus opens the door for using this unexplored dimeric structure space for many biological applications.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Peptide heterodimers are prevalent in nature, which are not only functional macromolecules but molecular tools for chemical and synthetic biology. Computational methods have also been developed to design heterodimers of advanced functions. However, these peptide heterodimers are usually formed through noncovalent interactions, which are prone to dissociate and subject to concentration-dependent nonspecific aggregation. Heterodimers crosslinked with interchain disulfide bonds are more stable, but it represents a formidable challenge for both the computational design of heterodimers and the manipulation of disulfide pairing for heterodimer synthesis and applications. Here, we report the design, synthesis and application of interchain disulfide-bridged peptide heterodimers with mutual orthogonality by combining computational de novo designs with a directed disulfide pairing strategy. These heterodimers can be used as not only scaffolds for generating functional molecules but chemical tools or building blocks for protein labeling and construction of crosslinking hybrids. This study thus opens the door for using this unexplored dimeric structure space for many biological applications.
2021
FROM THE LAB
Hosseinzadeh, Parisa and Watson, Paris R. and Craven, Timothy W. and Li, Xinting and Rettie, Stephen and Pardo-Avila, Fátima and Bera, Asim K. and Mulligan, Vikram Khipple and Lu, Peilong and Ford, Alexander S. and Weitzner, Brian D. and Stewart, Lance J. and Moyer, Adam P. and Di Piazza, Maddalena and Whalen, Joshua G. and Greisen, Per Jr. and Christianson, David W. and Baker, David
Anchor extension: a structure-guided approach to design cyclic peptides targeting enzyme active sites Journal Article
In: Nature Communications, 2021.
@article{Hosseinzadeh2021,
title = {Anchor extension: a structure-guided approach to design cyclic peptides targeting enzyme active sites},
author = {Hosseinzadeh, Parisa
and Watson, Paris R.
and Craven, Timothy W.
and Li, Xinting
and Rettie, Stephen
and Pardo-Avila, Fátima
and Bera, Asim K.
and Mulligan, Vikram Khipple
and Lu, Peilong
and Ford, Alexander S.
and Weitzner, Brian D.
and Stewart, Lance J.
and Moyer, Adam P.
and Di Piazza, Maddalena
and Whalen, Joshua G.
and Greisen, Per Jr.
and Christianson, David W.
and Baker, David},
url = {https://www.nature.com/articles/s41467-021-23609-8, Nature Communications
https://www.bakerlab.org/wp-content/uploads/2021/06/Hosseinzadeh_etal_NatureComms2021_AnchorExtention.pdf, Download PDF},
doi = {10.1038/s41467-021-23609-8},
year = {2021},
date = {2021-06-07},
urldate = {2021-06-07},
journal = {Nature Communications},
abstract = {Despite recent success in computational design of structured cyclic peptides, de novo design of cyclic peptides that bind to any protein functional site remains difficult. To address this challenge, we develop a computational “anchor extension” methodology for targeting protein interfaces by extending a peptide chain around a non-canonical amino acid residue anchor. To test our approach using a well characterized model system, we design cyclic peptides that inhibit histone deacetylases 2 and 6 (HDAC2 and HDAC6) with enhanced potency compared to the original anchor (IC50 values of 9.1 and 4.4 nM for the best binders compared to 5.4 and 0.6 µM for the anchor, respectively). The HDAC6 inhibitor is among the most potent reported so far. These results highlight the potential for de novo design of high-affinity protein-peptide interfaces, as well as the challenges that remain.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Despite recent success in computational design of structured cyclic peptides, de novo design of cyclic peptides that bind to any protein functional site remains difficult. To address this challenge, we develop a computational “anchor extension” methodology for targeting protein interfaces by extending a peptide chain around a non-canonical amino acid residue anchor. To test our approach using a well characterized model system, we design cyclic peptides that inhibit histone deacetylases 2 and 6 (HDAC2 and HDAC6) with enhanced potency compared to the original anchor (IC50 values of 9.1 and 4.4 nM for the best binders compared to 5.4 and 0.6 µM for the anchor, respectively). The HDAC6 inhibitor is among the most potent reported so far. These results highlight the potential for de novo design of high-affinity protein-peptide interfaces, as well as the challenges that remain.
Mulligan, Vikram Khipple, Workman, Sean, Sun, Tianjun, Rettie, Stephen, Li, Xinting, Worrall, Liam J., Craven, Timothy W., King, Dustin T., Hosseinzadeh, Parisa, Watkins, Andrew M., Renfrew, P. Douglas, Guffy, Sharon, Labonte, Jason W., Moretti, Rocco, Bonneau, Richard, Strynadka, Natalie C. J., Baker, David
Computationally designed peptide macrocycle inhibitors of New Delhi metallo-β-lactamase 1 Journal Article
In: Proceedings of the National Academy of Sciences, vol. 118, no. 12, 2021.
@article{Mulligan2021,
title = {Computationally designed peptide macrocycle inhibitors of New Delhi metallo-β-lactamase 1},
author = {Mulligan, Vikram Khipple and Workman, Sean and Sun, Tianjun and Rettie, Stephen and Li, Xinting and Worrall, Liam J. and Craven, Timothy W. and King, Dustin T. and Hosseinzadeh, Parisa and Watkins, Andrew M. and Renfrew, P. Douglas and Guffy, Sharon and Labonte, Jason W. and Moretti, Rocco and Bonneau, Richard and Strynadka, Natalie C. J. and Baker, David},
url = {https://www.pnas.org/content/118/12/e2012800118.full, PNAS
https://www.bakerlab.org/wp-content/uploads/2021/03/Mulligen_etal_PNAS2021_Macrocycle_inhibitors.pdf, Download PDF},
doi = {10.1073/pnas.2012800118},
year = {2021},
date = {2021-03-23},
urldate = {2021-03-23},
journal = {Proceedings of the National Academy of Sciences},
volume = {118},
number = {12},
abstract = {The rise of antibiotic resistance calls for new therapeutics targeting resistance factors such as the New Delhi metallo-β-lactamase 1 (NDM-1), a bacterial enzyme that degrades β-lactam antibiotics. We present structure-guided computational methods for designing peptide macrocycles built from mixtures of L- and D-amino acids that are able to bind to and inhibit targets of therapeutic interest. Our methods explicitly consider the propensity of a peptide to favor a binding-competent conformation, which we found to predict rank order of experimentally observed IC50 values across seven designed NDM-1- inhibiting peptides. We were able to determine X-ray crystal structures of three of the designed inhibitors in complex with NDM-1, and in all three the conformation of the peptide is very close to the computationally designed model. In two of the three structures, the binding mode with NDM-1 is also very similar to the design model, while in the third, we observed an alternative binding mode likely arising from internal symmetry in the shape of the design combined with flexibility of the target. Although challenges remain in robustly predicting target backbone changes, binding mode, and the effects of mutations on binding affinity, our methods for designing ordered, binding-competent macrocycles should have broad applicability to a wide range of therapeutic targets.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The rise of antibiotic resistance calls for new therapeutics targeting resistance factors such as the New Delhi metallo-β-lactamase 1 (NDM-1), a bacterial enzyme that degrades β-lactam antibiotics. We present structure-guided computational methods for designing peptide macrocycles built from mixtures of L- and D-amino acids that are able to bind to and inhibit targets of therapeutic interest. Our methods explicitly consider the propensity of a peptide to favor a binding-competent conformation, which we found to predict rank order of experimentally observed IC50 values across seven designed NDM-1- inhibiting peptides. We were able to determine X-ray crystal structures of three of the designed inhibitors in complex with NDM-1, and in all three the conformation of the peptide is very close to the computationally designed model. In two of the three structures, the binding mode with NDM-1 is also very similar to the design model, while in the third, we observed an alternative binding mode likely arising from internal symmetry in the shape of the design combined with flexibility of the target. Although challenges remain in robustly predicting target backbone changes, binding mode, and the effects of mutations on binding affinity, our methods for designing ordered, binding-competent macrocycles should have broad applicability to a wide range of therapeutic targets.
Quijano-Rubio, Alfredo and Yeh, Hsien-Wei and Park, Jooyoung and Lee, Hansol and Langan, Robert A. and Boyken, Scott E. and Lajoie, Marc J. and Cao, Longxing and Chow, Cameron M. and Miranda, Marcos C. and Wi, Jimin and Hong, Hyo Jeong and Stewart, Lance and Oh, Byung-Ha and Baker, David
De novo design of modular and tunable protein biosensors Journal Article
In: Nature, 2021.
@article{Quijano-Rubio2021,
title = {De novo design of modular and tunable protein biosensors},
author = {Quijano-Rubio, Alfredo
and Yeh, Hsien-Wei
and Park, Jooyoung
and Lee, Hansol
and Langan, Robert A.
and Boyken, Scott E.
and Lajoie, Marc J.
and Cao, Longxing
and Chow, Cameron M.
and Miranda, Marcos C.
and Wi, Jimin
and Hong, Hyo Jeong
and Stewart, Lance
and Oh, Byung-Ha
and Baker, David},
url = {https://www.nature.com/articles/s41586-021-03258-z, Nature
https://www.bakerlab.org/wp-content/uploads/2021/02/Rubio_et_al_Nature_COVID_LOCKR_sensors.pdf, Download PDF},
doi = {10.1038/s41586-021-03258-z},
year = {2021},
date = {2021-01-27},
urldate = {2021-01-27},
journal = {Nature},
abstract = {Naturally occurring protein switches have been repurposed for developing novel biosensors and reporters for cellular and clinical applications1, but the number of such switches is limited, and engineering them is often challenging as each is different. Here, we show that a very general class of protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which binding of a peptide key triggers biological outputs of interest2. The designed sensors are modular molecular devices with a closed dark state and an open luminescent state; binding of the analyte of interest drives switching from the closed to the open state. Because the sensor is based purely on thermodynamic coupling of analyte binding to sensor activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We demonstrate the modularity of this platform by creating biosensors that, with little optimization, sensitively detect the anti-apoptosis protein Bcl-2, the IgG1 Fc domain, the Her2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac Troponin I and an anti-Hepatitis B virus (HBV) antibody that achieve the sub-nanomolar sensitivity necessary to detect clinically relevant concentrations of these molecules. Given the current need for diagnostic tools for tracking COVID-193, we used the approach to design sensors of antibodies against SARS-CoV-2 protein epitopes and of the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. The latter, which incorporates a de novo designed RBD binder4, has a limit of detection of 15 pM and a signal over background of over 50-fold. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Naturally occurring protein switches have been repurposed for developing novel biosensors and reporters for cellular and clinical applications1, but the number of such switches is limited, and engineering them is often challenging as each is different. Here, we show that a very general class of protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which binding of a peptide key triggers biological outputs of interest2. The designed sensors are modular molecular devices with a closed dark state and an open luminescent state; binding of the analyte of interest drives switching from the closed to the open state. Because the sensor is based purely on thermodynamic coupling of analyte binding to sensor activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We demonstrate the modularity of this platform by creating biosensors that, with little optimization, sensitively detect the anti-apoptosis protein Bcl-2, the IgG1 Fc domain, the Her2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac Troponin I and an anti-Hepatitis B virus (HBV) antibody that achieve the sub-nanomolar sensitivity necessary to detect clinically relevant concentrations of these molecules. Given the current need for diagnostic tools for tracking COVID-193, we used the approach to design sensors of antibodies against SARS-CoV-2 protein epitopes and of the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. The latter, which incorporates a de novo designed RBD binder4, has a limit of detection of 15 pM and a signal over background of over 50-fold. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.
COLLABORATOR LED
Sorry, no publications matched your criteria.
2020
FROM THE LAB
Vikram Khipple Mulligan, Christine S. Kang, Michael R. Sawaya, Stephen Rettie, Xinting Li, Inna Antselovich, Timothy W. Craven, Andrew M. Watkins, Jason W. Labonte, Frank DiMaio, Todd O. Yeates, David Baker
Computational design of mixed chirality peptide macrocycles with internal symmetry Journal Article
In: Protein Science, 2020.
@article{Mulligan2020,
title = {Computational design of mixed chirality peptide macrocycles with internal symmetry},
author = {Vikram Khipple Mulligan and Christine S. Kang and Michael R. Sawaya and Stephen Rettie and Xinting Li and Inna Antselovich and Timothy W. Craven and Andrew M. Watkins and Jason W. Labonte and Frank DiMaio and Todd O. Yeates and David Baker},
url = {https://onlinelibrary.wiley.com/doi/epdf/10.1002/pro.3974
https://www.bakerlab.org/wp-content/uploads/2020/10/Mulligan2020-Computational-design-of-mixed-chirality-peptide-macrocycles-with-internal-symmetry.pdf},
doi = {10.1002/pro.3974},
year = {2020},
date = {2020-10-15},
journal = {Protein Science},
abstract = {Cyclic symmetry is frequent in protein and peptide homo‐oligomers, but extremely rare within a single chain, as it is not compatible with free N‐ and C‐termini. Here we describe the computational design of mixed‐chirality peptide macrocycles with rigid structures that feature internal cyclic symmetries or improper rotational symmetries inaccessible to natural proteins. Crystal structures of three C2‐ and C3‐symmetric macrocycles, and of six diverse S2‐symmetric macrocycles, match the computationally‐designed models with backbone heavy‐atom RMSD values of 1 å or better. Crystal structures of an S4‐symmetric macrocycle (consisting of a sequence and structure segment mirrored at each of three successive repeats) designed to bind zinc reveal a large‐scale zinc‐driven conformational change from an S4‐symmetric apo‐state to a nearly inverted S4‐symmetric holo‐state almost identical to the design model. This work demonstrates the power of computational design for exploring symmetries and structures not found in nature, and for creating synthetic switchable systems.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Cyclic symmetry is frequent in protein and peptide homo‐oligomers, but extremely rare within a single chain, as it is not compatible with free N‐ and C‐termini. Here we describe the computational design of mixed‐chirality peptide macrocycles with rigid structures that feature internal cyclic symmetries or improper rotational symmetries inaccessible to natural proteins. Crystal structures of three C2‐ and C3‐symmetric macrocycles, and of six diverse S2‐symmetric macrocycles, match the computationally‐designed models with backbone heavy‐atom RMSD values of 1 å or better. Crystal structures of an S4‐symmetric macrocycle (consisting of a sequence and structure segment mirrored at each of three successive repeats) designed to bind zinc reveal a large‐scale zinc‐driven conformational change from an S4‐symmetric apo‐state to a nearly inverted S4‐symmetric holo‐state almost identical to the design model. This work demonstrates the power of computational design for exploring symmetries and structures not found in nature, and for creating synthetic switchable systems.
Chunfu Xu, Peilong Lu, Tamer M. Gamal El-Din, Xue Y. Pei, Matthew C. Johnson, Atsuko Uyeda, Matthew J. Bick, Qi Xu, Daohua Jiang, Hua Bai, Gabriella Reggiano, Yang Hsia, T J Brunette, Jiayi Dou, Dan Ma, Eric M. Lynch, Scott E. Boyken, Po-Ssu Huang, Lance Stewart, Frank DiMaio, Justin M. Kollman, Ben F. Luisi, Tomoaki Matsuura, William A. Catterall, David Baker
Computational design of transmembrane pores Journal Article
In: Nature, vol. 585, pp. 129–134, 2020.
@article{Xu2020,
title = {Computational design of transmembrane pores},
author = {Chunfu Xu and Peilong Lu and Tamer M. Gamal El-Din and Xue Y. Pei and Matthew C. Johnson and Atsuko Uyeda and Matthew J. Bick and Qi Xu and Daohua Jiang and Hua Bai and Gabriella Reggiano and Yang Hsia and T J Brunette and Jiayi Dou and Dan Ma and Eric M. Lynch and Scott E. Boyken and Po-Ssu Huang and Lance Stewart and Frank DiMaio and Justin M. Kollman and Ben F. Luisi and Tomoaki Matsuura and William A. Catterall and David Baker },
url = {https://www.bakerlab.org/wp-content/uploads/2020/08/Xuetal_Nature2020_DeNovoPores.pdf
https://www.nature.com/articles/s41586-020-2646-5},
doi = {10.1038/s41586-020-2646-5},
year = {2020},
date = {2020-08-26},
journal = {Nature},
volume = {585},
pages = {129–134},
abstract = {Transmembrane channels and pores have key roles in fundamental biological processes and in biotechnological applications such as DNA nanopore sequencing, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels, and there have been recent advances in de novo membrane protein design and in redesigning naturally occurring channel-containing proteins. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore—but not the 12-helix pore—enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Transmembrane channels and pores have key roles in fundamental biological processes and in biotechnological applications such as DNA nanopore sequencing, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels, and there have been recent advances in de novo membrane protein design and in redesigning naturally occurring channel-containing proteins. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore—but not the 12-helix pore—enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.
COLLABORATOR LED
Robin L. Kirkpatrick, Kieran Lewis, Robert A. Langan, Marc J. Lajoie, Scott E. Boyken, Madeleine Eakman, David Baker, Jesse G. Zalatan
Conditional Recruitment to a DNA-Bound CRISPR–Cas Complex Using a Colocalization-Dependent Protein Switch Journal Article
In: ACS Synthetic Biology, 2020.
@article{Kirkpatrick2020,
title = {Conditional Recruitment to a DNA-Bound CRISPR–Cas Complex Using a Colocalization-Dependent Protein Switch},
author = {Robin L. Kirkpatrick and Kieran Lewis and Robert A. Langan and Marc J. Lajoie and Scott E. Boyken and Madeleine Eakman and David Baker and Jesse G. Zalatan},
url = {https://pubs.acs.org/doi/full/10.1021/acssynbio.0c00012
https://www.bakerlab.org/wp-content/uploads/2020/08/Kirkpatrick2020-LOCKR-CRISPR.pdf},
doi = {10.1021/acssynbio.0c00012},
year = {2020},
date = {2020-08-20},
journal = {ACS Synthetic Biology},
abstract = {To spatially control biochemical functions at specific sites within a genome, we have engineered a synthetic switch that activates when bound to its DNA target site. The system uses two CRISPR–Cas complexes to colocalize components of a de novo-designed protein switch (Co-LOCKR) to adjacent sites in the genome. Colocalization triggers a conformational change in the switch from an inactive closed state to an active open state with an exposed functional peptide. We prototype the system in yeast and demonstrate that DNA binding triggers activation of the switch, recruitment of a transcription factor, and expression of a downstream reporter gene. This DNA-triggered Co-LOCKR switch provides a platform to engineer sophisticated functions that should only be executed at a specific target site within the genome, with potential applications in a wide range of synthetic systems including epigenetic regulation, imaging, and genetic logic circuits.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
To spatially control biochemical functions at specific sites within a genome, we have engineered a synthetic switch that activates when bound to its DNA target site. The system uses two CRISPR–Cas complexes to colocalize components of a de novo-designed protein switch (Co-LOCKR) to adjacent sites in the genome. Colocalization triggers a conformational change in the switch from an inactive closed state to an active open state with an exposed functional peptide. We prototype the system in yeast and demonstrate that DNA binding triggers activation of the switch, recruitment of a transcription factor, and expression of a downstream reporter gene. This DNA-triggered Co-LOCKR switch provides a platform to engineer sophisticated functions that should only be executed at a specific target site within the genome, with potential applications in a wide range of synthetic systems including epigenetic regulation, imaging, and genetic logic circuits.
2019
FROM THE LAB
Langan, Robert A. , Boyken, Scott E. , Ng, Andrew H. , Samson, Jennifer A. , Dods, Galen , Westbrook, Alexandra M. , Nguyen, Taylor H. , Lajoie, Marc J. , Chen, Zibo , Berger, Stephanie , Mulligan, Vikram Khipple , Dueber, John E. , Novak, Walter R. P. , El-Samad, Hana , Baker, David
De novo design of bioactive protein switches Journal Article
In: Nature, 2019.
@article{Langan2019,
title = {De novo design of bioactive protein switches},
author = {Langan, Robert A.
and Boyken, Scott E.
and Ng, Andrew H.
and Samson, Jennifer A.
and Dods, Galen
and Westbrook, Alexandra M.
and Nguyen, Taylor H.
and Lajoie, Marc J.
and Chen, Zibo
and Berger, Stephanie
and Mulligan, Vikram Khipple
and Dueber, John E.
and Novak, Walter R. P.
and El-Samad, Hana
and Baker, David},
url = {https://doi.org/10.1038/s41586-019-1432-8
https://www.nature.com/articles/s41586-019-1432-8
https://www.bakerlab.org/wp-content/uploads/2019/07/Langan_LOCKR.pdf},
doi = {10.1038/s41586-019-1432-8},
year = {2019},
date = {2019-07-24},
journal = {Nature},
abstract = {Allosteric regulation of protein function is widespread in biology, but is challenging for de novo protein design as it requires the explicit design of multiple states with comparable free energies. Here we explore the possibility of designing switchable protein systems de novo, through the modulation of competing inter- and intramolecular interactions. We design a static, five-helix ‘cage’ with a single interface that can interact either intramolecularly with a terminal ‘latch’ helix or intermolecularly with a peptide ‘key’. Encoded on the latch are functional motifs for binding, degradation or nuclear export that function only when the key displaces the latch from the cage. We describe orthogonal cage–key systems that function in vitro, in yeast and in mammalian cells with up to 40-fold activation of function by key. The ability to design switchable protein functions that are controlled by induced conformational change is a milestone for de novo protein design, and opens up new avenues for synthetic biology and cell engineering.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Allosteric regulation of protein function is widespread in biology, but is challenging for de novo protein design as it requires the explicit design of multiple states with comparable free energies. Here we explore the possibility of designing switchable protein systems de novo, through the modulation of competing inter- and intramolecular interactions. We design a static, five-helix ‘cage’ with a single interface that can interact either intramolecularly with a terminal ‘latch’ helix or intermolecularly with a peptide ‘key’. Encoded on the latch are functional motifs for binding, degradation or nuclear export that function only when the key displaces the latch from the cage. We describe orthogonal cage–key systems that function in vitro, in yeast and in mammalian cells with up to 40-fold activation of function by key. The ability to design switchable protein functions that are controlled by induced conformational change is a milestone for de novo protein design, and opens up new avenues for synthetic biology and cell engineering.
Ng, Andrew H. and Nguyen, Taylor H. and Gómez-Schiavon, Mariana and Dods, Galen and Langan, Robert A. and Boyken, Scott E. and Samson, Jennifer A. and Waldburger, Lucas M. and Dueber, John E. and Baker, David and El-Samad, Hana
Modular and tunable biological feedback control using a de novo protein switch Journal Article
In: Nature, 2019.
@article{Ng2019,
title = {Modular and tunable biological feedback control using a de novo protein switch},
author = {Ng, Andrew H.
and Nguyen, Taylor H.
and Gómez-Schiavon, Mariana
and Dods, Galen
and Langan, Robert A.
and Boyken, Scott E.
and Samson, Jennifer A.
and Waldburger, Lucas M.
and Dueber, John E.
and Baker, David
and El-Samad, Hana},
url = {https://doi.org/10.1038/s41586-019-1425-7
https://www.nature.com/articles/s41586-019-1425-7
https://www.bakerlab.org/wp-content/uploads/2019/07/Ng_LOCKR_circuits.pdf},
doi = {10.1038/s41586-019-1425-7},
year = {2019},
date = {2019-07-24},
journal = {Nature},
abstract = {De novo-designed proteins1–3 hold great promise as building blocks for synthetic circuits, and can complement the use of engineered variants of natural proteins4–7. One such designer protein—degronLOCKR, which is based on ‘latching orthogonal cage–key proteins’ (LOCKR) technology8—is a switch that degrades a protein of interest in vivo upon induction by a genetically encoded small peptide. Here we leverage the plug-and-play nature of degronLOCKR to implement feedback control of endogenous signalling pathways and synthetic gene circuits. We first generate synthetic negative and positive feedback in the yeast mating pathway by fusing degronLOCKR to endogenous signalling molecules, illustrating the ease with which this strategy can be used to rewire complex endogenous pathways. We next evaluate feedback control mediated by degronLOCKR on a synthetic gene circuit9, to quantify the feedback capabilities and operational range of the feedback control circuit. The designed nature of degronLOCKR proteins enables simple and rational modifications to tune feedback behaviour in both the synthetic circuit and the mating pathway. The ability to engineer feedback control into living cells represents an important milestone in achieving the full potential of synthetic biology10,11,12. More broadly, this work demonstrates the large and untapped potential of de novo design of proteins for generating tools that implement complex synthetic functionalities in cells for biotechnological and therapeutic applications.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
De novo-designed proteins1–3 hold great promise as building blocks for synthetic circuits, and can complement the use of engineered variants of natural proteins4–7. One such designer protein—degronLOCKR, which is based on ‘latching orthogonal cage–key proteins’ (LOCKR) technology8—is a switch that degrades a protein of interest in vivo upon induction by a genetically encoded small peptide. Here we leverage the plug-and-play nature of degronLOCKR to implement feedback control of endogenous signalling pathways and synthetic gene circuits. We first generate synthetic negative and positive feedback in the yeast mating pathway by fusing degronLOCKR to endogenous signalling molecules, illustrating the ease with which this strategy can be used to rewire complex endogenous pathways. We next evaluate feedback control mediated by degronLOCKR on a synthetic gene circuit9, to quantify the feedback capabilities and operational range of the feedback control circuit. The designed nature of degronLOCKR proteins enables simple and rational modifications to tune feedback behaviour in both the synthetic circuit and the mating pathway. The ability to engineer feedback control into living cells represents an important milestone in achieving the full potential of synthetic biology10,11,12. More broadly, this work demonstrates the large and untapped potential of de novo design of proteins for generating tools that implement complex synthetic functionalities in cells for biotechnological and therapeutic applications.
COLLABORATOR LED
Sorry, no publications matched your criteria.
2018
FROM THE LAB
Shen, Hao, Fallas, Jorge A., Lynch, Eric, Sheffler, William, Parry, Bradley, Jannetty, Nicholas, Decarreau, Justin, Wagenbach, Michael, Vicente, Juan Jesus, Chen, Jiajun, Wang, Lei, Dowling, Quinton, Oberdorfer, Gustav, Stewart, Lance, Wordeman, Linda, De Yoreo, James, Jacobs-Wagner, Christine, Kollman, Justin, Baker, David
De novo design of self-assembling helical protein filaments Journal Article
In: Science, vol. 362, no. 6415, pp. 705–709, 2018, ISSN: 0036-8075.
@article{Shen2018,
title = {De novo design of self-assembling helical protein filaments},
author = {Shen, Hao and Fallas, Jorge A. and Lynch, Eric and Sheffler, William and Parry, Bradley and Jannetty, Nicholas and Decarreau, Justin and Wagenbach, Michael and Vicente, Juan Jesus and Chen, Jiajun and Wang, Lei and Dowling, Quinton and Oberdorfer, Gustav and Stewart, Lance and Wordeman, Linda and De Yoreo, James and Jacobs-Wagner, Christine and Kollman, Justin and Baker, David},
url = {http://science.sciencemag.org/content/362/6415/705
https://www.bakerlab.org/wp-content/uploads/2018/12/Shen2018_filaments.pdf},
doi = {10.1126/science.aau3775},
issn = {0036-8075},
year = {2018},
date = {2018-11-09},
journal = {Science},
volume = {362},
number = {6415},
pages = {705–709},
abstract = {There has been some success in designing stable peptide filaments; however, mimicking the reversible assembly of many natural protein filaments is challenging. Dynamic filaments usually comprise independently folded and asymmetric proteins and using such building blocks requires the design of multiple intermonomer interfaces. Shen et al. report the design of self-assembling helical filaments based on previously designed stable repeat proteins. The filaments are micron scale, and their diameter can be tuned by varying the number of repeats in the monomer. Anchor and capping units, built from monomers that lack an interaction interface, can be used to control assembly and disassembly.Science, this issue p. 705We describe a general computational approach to designing self-assembling helical filaments from monomeric proteins and use this approach to design proteins that assemble into micrometer-scale filaments with a wide range of geometries in vivo and in vitro. Cryo{textendash}electron microscopy structures of six designs are close to the computational design models. The filament building blocks are idealized repeat proteins, and thus the diameter of the filaments can be systematically tuned by varying the number of repeat units. The assembly and disassembly of the filaments can be controlled by engineered anchor and capping units built from monomers lacking one of the interaction surfaces. The ability to generate dynamic, highly ordered structures that span micrometers from protein monomers opens up possibilities for the fabrication of new multiscale metamaterials.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
There has been some success in designing stable peptide filaments; however, mimicking the reversible assembly of many natural protein filaments is challenging. Dynamic filaments usually comprise independently folded and asymmetric proteins and using such building blocks requires the design of multiple intermonomer interfaces. Shen et al. report the design of self-assembling helical filaments based on previously designed stable repeat proteins. The filaments are micron scale, and their diameter can be tuned by varying the number of repeats in the monomer. Anchor and capping units, built from monomers that lack an interaction interface, can be used to control assembly and disassembly.Science, this issue p. 705We describe a general computational approach to designing self-assembling helical filaments from monomeric proteins and use this approach to design proteins that assemble into micrometer-scale filaments with a wide range of geometries in vivo and in vitro. Cryo{textendash}electron microscopy structures of six designs are close to the computational design models. The filament building blocks are idealized repeat proteins, and thus the diameter of the filaments can be systematically tuned by varying the number of repeat units. The assembly and disassembly of the filaments can be controlled by engineered anchor and capping units built from monomers lacking one of the interaction surfaces. The ability to generate dynamic, highly ordered structures that span micrometers from protein monomers opens up possibilities for the fabrication of new multiscale metamaterials.
COLLABORATOR LED
Geiger-Schuller, Kathryn, Sforza, Kevin, Yuhas, Max, Parmeggiani, Fabio, Baker, David, Barrick, Doug
Extreme stability in de novo-designed repeat arrays is determined by unusually stable short-range interactions Journal Article
In: PNAS, vol. 115, no. 29, pp. 7539-7544, 2018, ISSN: 0027-8424.
@article{Geiger-Schuller2018,
title = {Extreme stability in de novo-designed repeat arrays is determined by unusually stable short-range interactions},
author = {Geiger-Schuller, Kathryn and Sforza, Kevin and Yuhas, Max and Parmeggiani, Fabio and Baker, David and Barrick, Doug},
url = {https://www.pnas.org/content/115/29/7539
https://www.bakerlab.org/wp-content/uploads/2019/02/Geiger-Schuller2018.pdf},
doi = {10.1073/pnas.1800283115},
issn = {0027-8424},
year = {2018},
date = {2018-07-17},
journal = {PNAS},
volume = {115},
number = {29},
pages = {7539-7544},
abstract = {We apply a statistical thermodynamic formalism to quantify the cooperativity of folding of de novo-designed helical repeat proteins (DHRs). This analysis provides a fundamental thermodynamic description of folding for de novo-designed proteins and permits comparison with naturally occurring repeat protein thermodynamics. We find that individual DHR units are intrinsically stable, unlike those of naturally occurring proteins. This observation reveals local (intrarepeat) interactions as a source of high stability in Rosetta-designed proteins and suggests that different types of DHR repeats may be combined in a single polypeptide chain, expanding the repertoire of folded DHRs for applications such as molecular recognition. Favorable intrinsic stability imparts a downhill shape to the energy landscape, suggesting that DHRs fold fast and through parallel pathways.Designed helical repeats (DHRs) are modular helix{textendash}loop{textendash}helix{textendash}loop protein structures that are tandemly repeated to form a superhelical array. Structures combining tandem DHRs demonstrate a wide range of molecular geometries, many of which are not observed in nature. Understanding cooperativity of DHR proteins provides insight into the molecular origins of Rosetta-based protein design hyperstability and facilitates comparison of energy distributions in artificial and naturally occurring protein folds. Here, we use a nearest-neighbor Ising model to quantify the intrinsic and interfacial free energies of four different DHRs. We measure the folding free energies of constructs with varying numbers of internal and terminal capping repeats for four different DHR folds, using guanidine-HCl and glycerol as destabilizing and solubilizing cosolvents. One-dimensional Ising analysis of these series reveals that, although interrepeat coupling energies are within the range seen for naturally occurring repeat proteins, the individual repeats of DHR proteins are intrinsically stable. This favorable intrinsic stability, which has not been observed for naturally occurring repeat proteins, adds to stabilizing interfaces, resulting in extraordinarily high stability. Stable repeats also impart a downhill shape to the energy landscape for DHR folding. These intrinsic stability differences suggest that part of the success of Rosetta-based design results from capturing favorable local interactions.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We apply a statistical thermodynamic formalism to quantify the cooperativity of folding of de novo-designed helical repeat proteins (DHRs). This analysis provides a fundamental thermodynamic description of folding for de novo-designed proteins and permits comparison with naturally occurring repeat protein thermodynamics. We find that individual DHR units are intrinsically stable, unlike those of naturally occurring proteins. This observation reveals local (intrarepeat) interactions as a source of high stability in Rosetta-designed proteins and suggests that different types of DHR repeats may be combined in a single polypeptide chain, expanding the repertoire of folded DHRs for applications such as molecular recognition. Favorable intrinsic stability imparts a downhill shape to the energy landscape, suggesting that DHRs fold fast and through parallel pathways.Designed helical repeats (DHRs) are modular helix{textendash}loop{textendash}helix{textendash}loop protein structures that are tandemly repeated to form a superhelical array. Structures combining tandem DHRs demonstrate a wide range of molecular geometries, many of which are not observed in nature. Understanding cooperativity of DHR proteins provides insight into the molecular origins of Rosetta-based protein design hyperstability and facilitates comparison of energy distributions in artificial and naturally occurring protein folds. Here, we use a nearest-neighbor Ising model to quantify the intrinsic and interfacial free energies of four different DHRs. We measure the folding free energies of constructs with varying numbers of internal and terminal capping repeats for four different DHR folds, using guanidine-HCl and glycerol as destabilizing and solubilizing cosolvents. One-dimensional Ising analysis of these series reveals that, although interrepeat coupling energies are within the range seen for naturally occurring repeat proteins, the individual repeats of DHR proteins are intrinsically stable. This favorable intrinsic stability, which has not been observed for naturally occurring repeat proteins, adds to stabilizing interfaces, resulting in extraordinarily high stability. Stable repeats also impart a downhill shape to the energy landscape for DHR folding. These intrinsic stability differences suggest that part of the success of Rosetta-based design results from capturing favorable local interactions.
2017-1988
ALL PAPERS
2017
Hosseinzadeh, Parisa*, Bhardwaj, Gaurav*, Mulligan, Vikram Khipple*, Shortridge, Matthew D., Craven, Timothy W., Pardo-Avila, F’atima, Rettie, Stephen A., Kim, David E., Silva, Daniel-Adriano, Ibrahim, Yehia M., Webb, Ian K., Cort, John R., Adkins, Joshua N., Varani, Gabriele, Baker, David
Comprehensive computational design of ordered peptide macrocycles Journal Article
In: Science, vol. 358, no. 6369, pp. 1461-1466, 2017, ISSN: 0036-8075.
@article{Hosseinzadeh2017,
title = {Comprehensive computational design of ordered peptide macrocycles},
author = {Hosseinzadeh, Parisa* and Bhardwaj, Gaurav* and Mulligan, Vikram Khipple* and Shortridge, Matthew D. and Craven, Timothy W. and Pardo-Avila, F{'a}tima and Rettie, Stephen A. and Kim, David E. and Silva, Daniel-Adriano and Ibrahim, Yehia M. and Webb, Ian K. and Cort, John R. and Adkins, Joshua N. and Varani, Gabriele and Baker, David},
url = {http://science.sciencemag.org/content/358/6369/1461
https://www.bakerlab.org/wp-content/uploads/2017/12/Science_Hosseinzadeh_et_al_2017.pdf},
doi = {10.1126/science.aap7577},
issn = {0036-8075},
year = {2017},
date = {2017-12-15},
journal = {Science},
volume = {358},
number = {6369},
pages = {1461-1466},
abstract = {Mixed-chirality peptide macrocycles such as cyclosporine are among the most potent therapeutics identified to date, but there is currently no way to systematically search the structural space spanned by such compounds. Natural proteins do not provide a useful guide: Peptide macrocycles lack regular secondary structures and hydrophobic cores, and can contain local structures not accessible with L-amino acids. Here, we enumerate the stable structures that can be adopted by macrocyclic peptides composed of L- and D-amino acids by near-exhaustive backbone sampling followed by sequence design and energy landscape calculations. We identify more than 200 designs predicted to fold into single stable structures, many times more than the number of currently available unbound peptide macrocycle structures. Nuclear magnetic resonance structures of 9 of 12 designed 7- to 10-residue macrocycles, and three 11- to 14-residue bicyclic designs, are close to the computational models. Our results provide a nearly complete coverage of the rich space of structures possible for short peptide macrocycles and vastly increase the available starting scaffolds for both rational drug design and library selection methods.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Mixed-chirality peptide macrocycles such as cyclosporine are among the most potent therapeutics identified to date, but there is currently no way to systematically search the structural space spanned by such compounds. Natural proteins do not provide a useful guide: Peptide macrocycles lack regular secondary structures and hydrophobic cores, and can contain local structures not accessible with L-amino acids. Here, we enumerate the stable structures that can be adopted by macrocyclic peptides composed of L- and D-amino acids by near-exhaustive backbone sampling followed by sequence design and energy landscape calculations. We identify more than 200 designs predicted to fold into single stable structures, many times more than the number of currently available unbound peptide macrocycle structures. Nuclear magnetic resonance structures of 9 of 12 designed 7- to 10-residue macrocycles, and three 11- to 14-residue bicyclic designs, are close to the computational models. Our results provide a nearly complete coverage of the rich space of structures possible for short peptide macrocycles and vastly increase the available starting scaffolds for both rational drug design and library selection methods.
Butterfield, Gabriel L.* and Lajoie, Marc J.* and Gustafson, Heather H. and Sellers, Drew L. and Nattermann, Una and Ellis, Daniel and Bale, Jacob B. and Ke, Sharon and Lenz, Garreck H. and Yehdego, Angelica and Ravichandran, Rashmi and Pun, Suzie H. and King, Neil P. and Baker, David
Evolution of a designed protein assembly encapsulating its own RNA genome Journal Article
In: Nature, 2017, ISSN: 1476-4687.
@article{Butterfield2017,
title = {Evolution of a designed protein assembly encapsulating its own RNA genome},
author = {Butterfield, Gabriel L.*
and Lajoie, Marc J.*
and Gustafson, Heather H.
and Sellers, Drew L.
and Nattermann, Una
and Ellis, Daniel
and Bale, Jacob B.
and Ke, Sharon
and Lenz, Garreck H.
and Yehdego, Angelica
and Ravichandran, Rashmi
and Pun, Suzie H.
and King, Neil P.
and Baker, David},
url = {http://dx.doi.org/10.1038/nature25157
https://www.bakerlab.org/wp-content/uploads/2017/12/Nature_Butterfield_et_al_2017.pdf},
doi = {10.1038/nature25157},
issn = {1476-4687},
year = {2017},
date = {2017-12-13},
journal = {Nature},
abstract = {The challenges of evolution in a complex biochemical environment, coupling genotype to phenotype and protecting the genetic material, are solved elegantly in biological systems by the encapsulation of nucleic acids. In the simplest examples, viruses use capsids to surround their genomes. Although these naturally occurring systems have been modified to change their tropism and to display proteins or peptides, billions of years of evolution have favoured efficiency at the expense of modularity, making viral capsids difficult to engineer. Synthetic systems composed of non-viral proteins could provide a ‘blank slate’ to evolve desired properties for drug delivery and other biomedical applications, while avoiding the safety risks and engineering challenges associated with viruses. Here we create synthetic nucleocapsids, which are computationally designed icosahedral protein assemblies with positively charged inner surfaces that can package their own full-length mRNA genomes. We explore the ability of these nucleocapsids to evolve virus-like properties by generating diversified populations using Escherichia coli as an expression host. Several generations of evolution resulted in markedly improved genome packaging (more than 133-fold), stability in blood (from less than 3.7% to 71% of packaged RNA protected after 6hours of treatment), and in vivo circulation time (from less than 5minutes to approximately 4.5hours). The resulting synthetic nucleocapsids package one full length RNA genome for every 11 icosahedral assemblies, similar to the best recombinant adeno-associated virus vectors. Our results show that there are simple evolutionary paths through which protein assemblies can acquire virus-like genome packaging and protection. Considerable effort has been directed at ‘top-down’ modification of viruses to be safe and effective for drug delivery and vaccine applications; the ability to design synthetic nanomaterials computationally and to optimize them through evolution now enables a complementary ‘bottom-up’ approach with considerable advantages in programmability and control.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The challenges of evolution in a complex biochemical environment, coupling genotype to phenotype and protecting the genetic material, are solved elegantly in biological systems by the encapsulation of nucleic acids. In the simplest examples, viruses use capsids to surround their genomes. Although these naturally occurring systems have been modified to change their tropism and to display proteins or peptides, billions of years of evolution have favoured efficiency at the expense of modularity, making viral capsids difficult to engineer. Synthetic systems composed of non-viral proteins could provide a ‘blank slate’ to evolve desired properties for drug delivery and other biomedical applications, while avoiding the safety risks and engineering challenges associated with viruses. Here we create synthetic nucleocapsids, which are computationally designed icosahedral protein assemblies with positively charged inner surfaces that can package their own full-length mRNA genomes. We explore the ability of these nucleocapsids to evolve virus-like properties by generating diversified populations using Escherichia coli as an expression host. Several generations of evolution resulted in markedly improved genome packaging (more than 133-fold), stability in blood (from less than 3.7% to 71% of packaged RNA protected after 6hours of treatment), and in vivo circulation time (from less than 5minutes to approximately 4.5hours). The resulting synthetic nucleocapsids package one full length RNA genome for every 11 icosahedral assemblies, similar to the best recombinant adeno-associated virus vectors. Our results show that there are simple evolutionary paths through which protein assemblies can acquire virus-like genome packaging and protection. Considerable effort has been directed at ‘top-down’ modification of viruses to be safe and effective for drug delivery and vaccine applications; the ability to design synthetic nanomaterials computationally and to optimize them through evolution now enables a complementary ‘bottom-up’ approach with considerable advantages in programmability and control.
2016
Gaurav Bhardwaj*, Vikram Khipple Mulligan*, Christopher D. Bahl*, Jason M. Gilmore, Peta J. Harvey, Olivier Cheneval, Garry W. Buchko, Surya V. S. R. K. Pulavarti, Quentin Kaas, Alexander Eletsky, Po-Ssu Huang, William A. Johnsen, Per Jr Greisen, Gabriel J. Rocklin, Yifan Song, Thomas W. Linsky, Andrew Watkins, Stephen A. Rettie, Xianzhong Xu, Lauren P. Carter, Richard Bonneau, James M. Olson, Evangelos Coutsias, Colin E. Correnti, Thomas Szyperski, David J. Craik, David Baker
Accurate de novo design of hyperstable constrained peptides Journal Article
In: Nature, 2016.
@article{Bhardwaj2016,
title = {Accurate de novo design of hyperstable constrained peptides},
author = { Gaurav Bhardwaj* and Vikram Khipple Mulligan* and Christopher D. Bahl* and Jason M. Gilmore and Peta J. Harvey and Olivier Cheneval and Garry W. Buchko and Surya V. S. R. K. Pulavarti and Quentin Kaas and Alexander Eletsky and Po-Ssu Huang and William A. Johnsen and Per Jr Greisen and Gabriel J. Rocklin and Yifan Song and Thomas W. Linsky and Andrew Watkins and Stephen A. Rettie and Xianzhong Xu, Lauren P. Carter and Richard Bonneau and James M. Olson and Evangelos Coutsias and Colin E. Correnti and Thomas Szyperski and David J. Craik and David Baker },
url = {https://www.bakerlab.org/wp-content/uploads/2016/09/Bhardwaj_Nature_2016.pdf},
doi = {10.1038/nature19791},
year = {2016},
date = {2016-09-14},
journal = {Nature},
abstract = {Naturally occurring, pharmacologically active peptides constrained with covalent crosslinks generally have shapes that have evolved to fit precisely into binding pockets on their targets. Such peptides can have excellent pharmaceutical properties, combining the stability and tissue penetration of small-molecule drugs with the specificity of much larger protein therapeutics. The ability to design constrained peptides with precisely specified tertiary structures would enable the design of shape-complementary inhibitors of arbitrary targets. Here we describe the development of computational methods for accurate de novo design of conformationally restricted peptides, and the use of these methods to design 18–47 residue, disulfide-crosslinked peptides, a subset of which are heterochiral and/or N–C backbone-cyclized. Both genetically encodable and non-canonical peptides are exceptionally stable to thermal and chemical denaturation, and 12 experimentally determined X-ray and NMR structures are nearly identical to the computational design models. The computational design methods and stable scaffolds presented here provide the basis for development of a new generation of peptide-based drugs.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Naturally occurring, pharmacologically active peptides constrained with covalent crosslinks generally have shapes that have evolved to fit precisely into binding pockets on their targets. Such peptides can have excellent pharmaceutical properties, combining the stability and tissue penetration of small-molecule drugs with the specificity of much larger protein therapeutics. The ability to design constrained peptides with precisely specified tertiary structures would enable the design of shape-complementary inhibitors of arbitrary targets. Here we describe the development of computational methods for accurate de novo design of conformationally restricted peptides, and the use of these methods to design 18–47 residue, disulfide-crosslinked peptides, a subset of which are heterochiral and/or N–C backbone-cyclized. Both genetically encodable and non-canonical peptides are exceptionally stable to thermal and chemical denaturation, and 12 experimentally determined X-ray and NMR structures are nearly identical to the computational design models. The computational design methods and stable scaffolds presented here provide the basis for development of a new generation of peptide-based drugs.
2015
L Doyle, J Hallinan, J Bolduc, F Parmeggiani, D Baker, BL Stoddard, P Bradley
Rational design of α-helical tandem repeat proteins with closed architectures Journal Article
In: Nature, vol. 528(7583), pp. 585-8, 2015.
@article{L2015,
title = {Rational design of α-helical tandem repeat proteins with closed architectures},
author = {L Doyle and J Hallinan and J Bolduc and F Parmeggiani and D Baker and BL Stoddard and P Bradley},
url = {https://www.bakerlab.org/wp-content/uploads/2015/12/Doyle_Nature_2015.pdf},
doi = {10.1038/nature16191},
year = {2015},
date = {2015-12-24},
journal = {Nature},
volume = {528(7583)},
pages = {585-8},
abstract = {Tandem repeat proteins, which are formed by repetition of modular units of protein sequence and structure, play important biological roles as macromolecular binding and scaffolding domains, enzymes, and building blocks for the assembly of fibrous materials. The modular nature of repeat proteins enables the rapid construction and diversification of extended binding surfaces by duplication and recombination of simple building blocks. The overall architecture of tandem repeat protein structures--which is dictated by the internal geometry and local packing of the repeat building blocks--is highly diverse, ranging from extended, super-helical folds that bind peptide, DNA, and RNA partners, to closed and compact conformations with internal cavities suitable for small molecule binding and catalysis. Here we report the development and validation of computational methods for de novo design of tandem repeat protein architectures driven purely by geometric criteria defining the inter-repeat geometry, without reference to the sequences and structures of existing repeat protein families. We have applied these methods to design a series of closed α-solenoid repeat structures (α-toroids) in which the inter-repeat packing geometry is constrained so as to juxtapose the amino (N) and carboxy (C) termini; several of these designed structures have been validated by X-ray crystallography. Unlike previous approaches to tandem repeat protein engineering, our design procedure does not rely on template sequence or structural information taken from natural repeat proteins and hence can produce structures unlike those seen in nature. As an example, we have successfully designed and validated closed α-solenoid repeats with a left-handed helical architecture that--to our knowledge--is not yet present in the protein structure database.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Tandem repeat proteins, which are formed by repetition of modular units of protein sequence and structure, play important biological roles as macromolecular binding and scaffolding domains, enzymes, and building blocks for the assembly of fibrous materials. The modular nature of repeat proteins enables the rapid construction and diversification of extended binding surfaces by duplication and recombination of simple building blocks. The overall architecture of tandem repeat protein structures–which is dictated by the internal geometry and local packing of the repeat building blocks–is highly diverse, ranging from extended, super-helical folds that bind peptide, DNA, and RNA partners, to closed and compact conformations with internal cavities suitable for small molecule binding and catalysis. Here we report the development and validation of computational methods for de novo design of tandem repeat protein architectures driven purely by geometric criteria defining the inter-repeat geometry, without reference to the sequences and structures of existing repeat protein families. We have applied these methods to design a series of closed α-solenoid repeat structures (α-toroids) in which the inter-repeat packing geometry is constrained so as to juxtapose the amino (N) and carboxy (C) termini; several of these designed structures have been validated by X-ray crystallography. Unlike previous approaches to tandem repeat protein engineering, our design procedure does not rely on template sequence or structural information taken from natural repeat proteins and hence can produce structures unlike those seen in nature. As an example, we have successfully designed and validated closed α-solenoid repeats with a left-handed helical architecture that–to our knowledge–is not yet present in the protein structure database.
TJ Brunette, F Parmeggiani, PS Huang, G Bhabha, DC Ekiert, SE Tsutakawa, GL Hura, JA Tainer, D Baker
Exploring the repeat protein universe through computational protein design Journal Article
In: Nature, vol. 528(7583), pp. 580-4, 2015.
@article{TJ2015,
title = {Exploring the repeat protein universe through computational protein design},
author = {TJ Brunette and F Parmeggiani and PS Huang and G Bhabha and DC Ekiert and SE Tsutakawa and GL Hura and JA Tainer and D Baker},
url = {https://www.bakerlab.org/wp-content/uploads/2015/12/Brunette_Nature_2015.pdf},
doi = {10.1038/nature16162},
year = {2015},
date = {2015-12-24},
journal = {Nature},
volume = {528(7583)},
pages = {580-4},
abstract = {A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes. Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications. Here we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix-loop-helix-loop structural motif. Eighty-three designs with sequences unrelated to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 °C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 Å. Our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering. },
keywords = {},
pubstate = {published},
tppubtype = {article}
}
A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes. Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications. Here we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix-loop-helix-loop structural motif. Eighty-three designs with sequences unrelated to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 °C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 Å. Our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering.
Clancey Wolf, Justin B Siegel, Christine Tinberg, Alessandra Camarca, Carmen Gianfrani, Shirley Paski, Rongjin Guan, Gaetano T Montelione, David Baker, Ingrid S Pultz
Engineering of Kuma030: a gliadin peptidase that rapidly degrades immunogenic gliadin peptides in gastric conditions. Journal Article
In: Journal of the American Chemical Society, 2015, ISSN: 1520-5126.
@article{617,
title = {Engineering of Kuma030: a gliadin peptidase that rapidly degrades immunogenic gliadin peptides in gastric conditions.},
author = { Clancey Wolf and Justin B Siegel and Christine Tinberg and Alessandra Camarca and Carmen Gianfrani and Shirley Paski and Rongjin Guan and Gaetano T Montelione and David Baker and Ingrid S Pultz},
url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Wolf_JACS_2015.pdf},
doi = {10.1021/jacs.5b08325},
issn = {1520-5126},
year = {2015},
date = {2015-09-01},
journal = {Journal of the American Chemical Society},
abstract = {Celiac disease is characterized by intestinal inflammation triggered by gliadin, a component of dietary gluten. Oral administration of proteases that can rapidly degrade gliadin in the gastric compartment has been proposed as a treatment for celiac disease; however, no protease has been shown to specifically reduce the immunogenic gliadin content, in gastric conditions, to below the threshold shown to be toxic for celiac patients. Here, we used the Rosetta Molecular Modeling Suite to redesign the active site of the acid-active gliadin endopeptidase KumaMax. The resulting protease, Kuma030, specifically recognizes tripeptide sequences that are found throughout the immunogenic regions of gliadin, as well as in homologous proteins in barley and rye. Indeed, treatment of gliadin with Kuma030 eliminates the ability of gliadin to stimulate a T cell response. Kuma030 is capable of degrading >99% of the immunogenic gliadin fraction in laboratory-simulated gastric digestions with minutes, to a level below the toxic threshold for celiac patients, suggesting great potential for this enzyme as an oral therapeutic for celiac disease.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Celiac disease is characterized by intestinal inflammation triggered by gliadin, a component of dietary gluten. Oral administration of proteases that can rapidly degrade gliadin in the gastric compartment has been proposed as a treatment for celiac disease; however, no protease has been shown to specifically reduce the immunogenic gliadin content, in gastric conditions, to below the threshold shown to be toxic for celiac patients. Here, we used the Rosetta Molecular Modeling Suite to redesign the active site of the acid-active gliadin endopeptidase KumaMax. The resulting protease, Kuma030, specifically recognizes tripeptide sequences that are found throughout the immunogenic regions of gliadin, as well as in homologous proteins in barley and rye. Indeed, treatment of gliadin with Kuma030 eliminates the ability of gliadin to stimulate a T cell response. Kuma030 is capable of degrading >99% of the immunogenic gliadin fraction in laboratory-simulated gastric digestions with minutes, to a level below the toxic threshold for celiac patients, suggesting great potential for this enzyme as an oral therapeutic for celiac disease.
E H Egelman, C. Xu, F. DiMaio, E Magnotti, C Modlin, X Yu, E Wright, D Baker, V P Conticello
Structural plasticity of helical nanotubes based on coiled-coil assemblies. Journal Article
In: Structure (London, England : 1993), vol. 23, pp. 280-9, 2015, ISSN: 1878-4186.
@article{609,
title = {Structural plasticity of helical nanotubes based on coiled-coil assemblies.},
author = { E H Egelman and C. Xu and F. DiMaio and E Magnotti and C Modlin and X Yu and E Wright and D Baker and V P Conticello},
url = {http://www.bakerlab.org/wp-content/uploads/2015/12/structuralplasticity_Baker2015.pdf},
doi = {10.1016/j.str.2014.12.008},
issn = {1878-4186},
year = {2015},
date = {2015-02-01},
journal = {Structure (London, England : 1993)},
volume = {23},
pages = {280-9},
abstract = {Numerous instances can be seen in evolution in which protein quaternary structures have diverged while the sequences of the building blocks have remained fairly conserved. However, the path through which such divergence has taken place is usually not known. We have designed two synthetic 29-residue α-helical peptides, based on the coiled-coil structural motif, that spontaneously self-assemble into helical nanotubes in vitro. Using electron cryomicroscopy with a newly available direct electron detection capability, we can achieve near-atomic resolution of these thin structures. We show how conservative changes of only one or two amino acids result in dramatic changes in quaternary structure, in which the assemblies can be switched between two very different forms. This system provides a framework for understanding how small sequence changes in evolution can translate into very large changes in supramolecular structure, a phenomenon that may have significant implications for the de novo design of synthetic peptide assemblies.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Numerous instances can be seen in evolution in which protein quaternary structures have diverged while the sequences of the building blocks have remained fairly conserved. However, the path through which such divergence has taken place is usually not known. We have designed two synthetic 29-residue α-helical peptides, based on the coiled-coil structural motif, that spontaneously self-assemble into helical nanotubes in vitro. Using electron cryomicroscopy with a newly available direct electron detection capability, we can achieve near-atomic resolution of these thin structures. We show how conservative changes of only one or two amino acids result in dramatic changes in quaternary structure, in which the assemblies can be switched between two very different forms. This system provides a framework for understanding how small sequence changes in evolution can translate into very large changes in supramolecular structure, a phenomenon that may have significant implications for the de novo design of synthetic peptide assemblies.
2014
Daniel S Liu, Lucas G Niv’on, Florian Richter, Peter J Goldman, Thomas J Deerinck, Jennifer Z Yao, Douglas Richardson, William S Phipps, Anne Z Ye, Mark H Ellisman, Catherine L Drennan, David Baker, Alice Y Ting
Computational design of a red fluorophore ligase for site-specific protein labeling in living cells. Journal Article
In: Proceedings of the National Academy of Sciences of the United States of America, vol. 111, pp. E4551-9, 2014, ISSN: 1091-6490.
@article{619,
title = {Computational design of a red fluorophore ligase for site-specific protein labeling in living cells.},
author = { Daniel S Liu and Lucas G Niv'on and Florian Richter and Peter J Goldman and Thomas J Deerinck and Jennifer Z Yao and Douglas Richardson and William S Phipps and Anne Z Ye and Mark H Ellisman and Catherine L Drennan and David Baker and Alice Y Ting},
url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Liu_computational_PNAS_2014.pdf},
doi = {10.1073/pnas.1404736111},
issn = {1091-6490},
year = {2014},
date = {2014-10-01},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {111},
pages = {E4551-9},
abstract = {Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.
Chris King, Esteban N Garza, Ronit Mazor, Jonathan L Linehan, Ira Pastan, Marion Pepper, David Baker
Removing T-cell epitopes with computational protein design. Journal Article
In: Proceedings of the National Academy of Sciences of the United States of America, 2014, ISSN: 1091-6490.
@article{533,
title = {Removing T-cell epitopes with computational protein design.},
author = { Chris King and Esteban N Garza and Ronit Mazor and Jonathan L Linehan and Ira Pastan and Marion Pepper and David Baker},
url = {http://www.bakerlab.org/wp-content/uploads/2015/12/King_PNAS_2014A.pdf},
doi = {10.1073/pnas.1321126111},
issn = {1091-6490},
year = {2014},
date = {2014-05-01},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
abstract = {Immune responses can make protein therapeutics ineffective or even dangerous. We describe a general computational protein design method for reducing immunogenicity by eliminating known and predicted T-cell epitopes and maximizing the content of human peptide sequences without disrupting protein structure and function. We show that the method recapitulates previous experimental results on immunogenicity reduction, and we use it to disrupt T-cell epitopes in GFP and Pseudomonas exotoxin A without disrupting function.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Immune responses can make protein therapeutics ineffective or even dangerous. We describe a general computational protein design method for reducing immunogenicity by eliminating known and predicted T-cell epitopes and maximizing the content of human peptide sequences without disrupting protein structure and function. We show that the method recapitulates previous experimental results on immunogenicity reduction, and we use it to disrupt T-cell epitopes in GFP and Pseudomonas exotoxin A without disrupting function.
Tyler D Shropshire, Jack Reifert, Sridharan Rajagopalan, David Baker, Stuart C Feinstein, Patrick S Daugherty
Amyloid β peptide cleavage by kallikrein 7 attenuates fibril growth and rescues neurons from Aβ-mediated toxicity in vitro. Journal Article
In: Biological chemistry, vol. 395, pp. 109-18, 2014, ISSN: 1437-4315.
@article{510,
title = {Amyloid β peptide cleavage by kallikrein 7 attenuates fibril growth and rescues neurons from Aβ-mediated toxicity in vitro.},
author = { Tyler D Shropshire and Jack Reifert and Sridharan Rajagopalan and David Baker and Stuart C Feinstein and Patrick S Daugherty},
doi = {10.1515/hsz-2013-0230},
issn = {1437-4315},
year = {2014},
date = {2014-01-01},
journal = {Biological chemistry},
volume = {395},
pages = {109-18},
abstract = {Abstract The gradual accumulation and assembly of β-amyloid (Aβ) peptide into neuritic plaques is a major pathological hallmark of Alzheimer disease (AD). Proteolytic degradation of Aβ is an important clearance mechanism under normal circumstances, and it has been found to be compromised in those with AD. Here, the extended substrate specificity and Aβ-degrading capacity of kallikrein 7 (KLK7), a serine protease with a unique chymotrypsin-like specificity, was characterized. Preferred peptide substrates of KLK7 identified using a bacterial display substrate library were found to exhibit a consensus motif of RXΦ(Y/F)textdownarrow(Y/F)textdownarrow(S/A/G/T) or RXΦ(Y/F)textdownarrow(S/T/A) (Φ=hydrophobic), which is remarkably similar to the hydrophobic core motif of Aβ (K16L17V18F19F20 A21) that is largely responsible for aggregation propensity. KLK7 was found to cleave after both Phe residues within the core of Aβ42 in vitro, thereby inhibiting Aβ fibril formation and promoting the degradation of preformed fibrils. Finally, the treatment of Aβ oligomer preparations with KLK7, but not inactive pro-KLK7, significantly reduced Aβ42-mediated toxicity to rat hippocampal neurons to the same extent as the known Aβ-degrading protease insulin-degrading enzyme (IDE). Taken together, these results indicate that KLK7 possesses an Aβ-degrading capacity that can ameliorate the toxic effects of the aggregated peptide in vitro.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Abstract The gradual accumulation and assembly of β-amyloid (Aβ) peptide into neuritic plaques is a major pathological hallmark of Alzheimer disease (AD). Proteolytic degradation of Aβ is an important clearance mechanism under normal circumstances, and it has been found to be compromised in those with AD. Here, the extended substrate specificity and Aβ-degrading capacity of kallikrein 7 (KLK7), a serine protease with a unique chymotrypsin-like specificity, was characterized. Preferred peptide substrates of KLK7 identified using a bacterial display substrate library were found to exhibit a consensus motif of RXΦ(Y/F)textdownarrow(Y/F)textdownarrow(S/A/G/T) or RXΦ(Y/F)textdownarrow(S/T/A) (Φ=hydrophobic), which is remarkably similar to the hydrophobic core motif of Aβ (K16L17V18F19F20 A21) that is largely responsible for aggregation propensity. KLK7 was found to cleave after both Phe residues within the core of Aβ42 in vitro, thereby inhibiting Aβ fibril formation and promoting the degradation of preformed fibrils. Finally, the treatment of Aβ oligomer preparations with KLK7, but not inactive pro-KLK7, significantly reduced Aβ42-mediated toxicity to rat hippocampal neurons to the same extent as the known Aβ-degrading protease insulin-degrading enzyme (IDE). Taken together, these results indicate that KLK7 possesses an Aβ-degrading capacity that can ameliorate the toxic effects of the aggregated peptide in vitro.
Pierre Leblanc, Leonard Moise, Cybelle Luza, Kanawat Chantaralawan, Lynchy Lezeau, Jianping Yuan, Mary Field, Daniel Richer, Christine Boyle, William D Martin, Jordan B Fishman, Eric A Berg, David Baker, Brandon Zeigler, Dale E Mais, William Taylor, Russell Coleman, H Shaw Warren, Jeffrey A Gelfand, Anne S De Groot, Timothy Brauns, Mark C Poznansky
VaxCelerate II: rapid development of a self-assembling vaccine for Lassa fever. Journal Article
In: Human vaccines & immunotherapeutics, vol. 10, pp. 3022-38, 2014, ISSN: 2164-554X.
@article{618,
title = {VaxCelerate II: rapid development of a self-assembling vaccine for Lassa fever.},
author = { Pierre Leblanc and Leonard Moise and Cybelle Luza and Kanawat Chantaralawan and Lynchy Lezeau and Jianping Yuan and Mary Field and Daniel Richer and Christine Boyle and William D Martin and Jordan B Fishman and Eric A Berg and David Baker and Brandon Zeigler and Dale E Mais and William Taylor and Russell Coleman and H Shaw Warren and Jeffrey A Gelfand and Anne S De Groot and Timothy Brauns and Mark C Poznansky},
url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Leblanc_HumanVI_2014.pdf},
doi = {10.4161/hv.34413},
issn = {2164-554X},
year = {2014},
date = {2014-00-01},
journal = {Human vaccines & immunotherapeutics},
volume = {10},
pages = {3022-38},
abstract = {Development of effective vaccines against emerging infectious diseases (EID) can take as much or more than a decade to progress from pathogen isolation/identification to clinical approval. As a result, conventional approaches fail to produce field-ready vaccines before the EID has spread extensively. Lassa is a prototypical emerging infectious disease endemic to West Africa for which no successful vaccine is available. We established the VaxCelerate Consortium to address the need for more rapid vaccine development by creating a platform capable of generating and pre-clinically testing a new vaccine against specific pathogen targets in less than 120 d A self-assembling vaccine is at the core of the approach. It consists of a fusion protein composed of the immunostimulatory Mycobacterium tuberculosis heat shock protein 70 (MtbHSP70) and the biotin binding protein, avidin. Mixing the resulting protein (MAV) with biotinylated pathogen-specific immunogenic peptides yields a self-assembled vaccine (SAV). To meet the time constraint imposed on this project, we used a distributed R&D model involving experts in the fields of protein engineering and production, bioinformatics, peptide synthesis/design and GMP/GLP manufacturing and testing standards. SAV immunogenicity was first tested using H1N1 influenza specific peptides and the entire VaxCelerate process was then tested in a mock live-fire exercise targeting Lassa fever virus. We demonstrated that the Lassa fever vaccine induced significantly increased class II peptide specific interferon-γ CD4(+) T cell responses in HLA-DR3 transgenic mice compared to peptide or MAV alone controls. We thereby demonstrated that our SAV in combination with a distributed development model may facilitate accelerated regulatory review by using an identical design for each vaccine and by applying safety and efficacy assessment tools that are more relevant to human vaccine responses than current animal models.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Development of effective vaccines against emerging infectious diseases (EID) can take as much or more than a decade to progress from pathogen isolation/identification to clinical approval. As a result, conventional approaches fail to produce field-ready vaccines before the EID has spread extensively. Lassa is a prototypical emerging infectious disease endemic to West Africa for which no successful vaccine is available. We established the VaxCelerate Consortium to address the need for more rapid vaccine development by creating a platform capable of generating and pre-clinically testing a new vaccine against specific pathogen targets in less than 120 d A self-assembling vaccine is at the core of the approach. It consists of a fusion protein composed of the immunostimulatory Mycobacterium tuberculosis heat shock protein 70 (MtbHSP70) and the biotin binding protein, avidin. Mixing the resulting protein (MAV) with biotinylated pathogen-specific immunogenic peptides yields a self-assembled vaccine (SAV). To meet the time constraint imposed on this project, we used a distributed R&D model involving experts in the fields of protein engineering and production, bioinformatics, peptide synthesis/design and GMP/GLP manufacturing and testing standards. SAV immunogenicity was first tested using H1N1 influenza specific peptides and the entire VaxCelerate process was then tested in a mock live-fire exercise targeting Lassa fever virus. We demonstrated that the Lassa fever vaccine induced significantly increased class II peptide specific interferon-γ CD4(+) T cell responses in HLA-DR3 transgenic mice compared to peptide or MAV alone controls. We thereby demonstrated that our SAV in combination with a distributed development model may facilitate accelerated regulatory review by using an identical design for each vaccine and by applying safety and efficacy assessment tools that are more relevant to human vaccine responses than current animal models.
2013
MA Molski, JL Goodman, FC Chou, D Baker, R Das, A Schepartz
Remodeling a beta-peptide bundle Journal Article
In: Chemical Science, vol. 4, pp. 319-324, 2013, ISSN: 2041-6520.
@article{605,
title = {Remodeling a beta-peptide bundle},
author = { MA Molski and JL Goodman and FC Chou and D Baker and R Das and A Schepartz},
url = {http://www.bakerlab.org/wp-content/uploads/2015/12/remodelingabeta_Baker2013.pdf},
doi = {10.1039/c2sc21117c},
issn = {2041-6520},
year = {2013},
date = {2013-00-01},
journal = {Chemical Science},
volume = {4},
pages = {319-324},
abstract = {Natural biopolymers fold with fidelity, burying diverse side chains into well-packed cores and protecting their backbones from solvent. Certain beta-peptide oligomers assemble into bundles of defined octameric stoichiometry that resemble natural proteins in many respects. These beta-peptide bundles are thermostable, fold cooperatively, exchange interior amide N-H protons slowly, exclude hydrophobic dyes, and can be characterized at high resolution using X-ray crystallography - just like many proteins found in nature. But unlike natural proteins, all octameric beta-peptide bundles contain a sequence-uniform hydrophobic core composed of 32 leucine side chains. Here we apply rational design principles, including the Rosetta computational design methodology, to introduce sequence diversity into the bundle core while retaining the characteristic beta-peptide bundle fold. Using circular dichroism spectroscopy and analytical ultracentrifugation, we confirmed the prediction that an octameric bundle still assembles upon a major remodelling of its core: the mutation of sixteen core beta-homo-leucine side chains into sixteen beta-homo-phenylalanine side chains. Nevertheless, the bundle containing a partially beta-homo-phenylalanine core poorly protects interior amide protons from exchange, suggesting molten-globule-like properties. We further improve stability by the incorporation of eight beta-homo-pentafluorophenyalanine side chains, giving an assembly with amide protection factors comparable to prior well-structured bundles. By demonstrating that their cores tolerate significant sequence variation, the beta-peptide bundles reported here represent a starting point for the "bottom-up" construction of beta-peptide assemblies possessing both structure and sophisticated function.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Natural biopolymers fold with fidelity, burying diverse side chains into well-packed cores and protecting their backbones from solvent. Certain beta-peptide oligomers assemble into bundles of defined octameric stoichiometry that resemble natural proteins in many respects. These beta-peptide bundles are thermostable, fold cooperatively, exchange interior amide N-H protons slowly, exclude hydrophobic dyes, and can be characterized at high resolution using X-ray crystallography – just like many proteins found in nature. But unlike natural proteins, all octameric beta-peptide bundles contain a sequence-uniform hydrophobic core composed of 32 leucine side chains. Here we apply rational design principles, including the Rosetta computational design methodology, to introduce sequence diversity into the bundle core while retaining the characteristic beta-peptide bundle fold. Using circular dichroism spectroscopy and analytical ultracentrifugation, we confirmed the prediction that an octameric bundle still assembles upon a major remodelling of its core: the mutation of sixteen core beta-homo-leucine side chains into sixteen beta-homo-phenylalanine side chains. Nevertheless, the bundle containing a partially beta-homo-phenylalanine core poorly protects interior amide protons from exchange, suggesting molten-globule-like properties. We further improve stability by the incorporation of eight beta-homo-pentafluorophenyalanine side chains, giving an assembly with amide protection factors comparable to prior well-structured bundles. By demonstrating that their cores tolerate significant sequence variation, the beta-peptide bundles reported here represent a starting point for the "bottom-up" construction of beta-peptide assemblies possessing both structure and sophisticated function.
2012
Sydney R Gordon, Elizabeth J Stanley, Sarah Wolf, Angus Toland, Sean J Wu, Daniel Hadidi, Jeremy H Mills, David Baker, Ingrid Swanson Pultz, Justin B Siegel
Computational Design of an α-gliadin Peptidase Journal Article
In: Journal of the American Chemical Society, vol. 134, pp. 20513-20, 2012, ISSN: 1520-5126.
@article{479,
title = {Computational Design of an α-gliadin Peptidase},
author = { Sydney R Gordon and Elizabeth J Stanley and Sarah Wolf and Angus Toland and Sean J Wu and Daniel Hadidi and Jeremy H Mills and David Baker and Ingrid Swanson Pultz and Justin B Siegel},
url = {https://www.bakerlab.org/wp-content/uploads/2015/12/Gordon12E.pdf
http://www.ncbi.nlm.nih.gov/pubmed/23153249},
doi = {10.1021/ja3094795},
issn = {1520-5126},
year = {2012},
date = {2012-12-01},
journal = {Journal of the American Chemical Society},
volume = {134},
pages = {20513-20},
abstract = {The ability to rationally modify enzymes to perform novel chemical transformations is essential for the rapid production of next-generation protein therapeutics. Here we describe the use of chemical principles to identify a naturally occurring acid-active peptidase, and the subsequent use of computational protein design tools to reengineer its specificity toward immunogenic elements found in gluten that are the proposed cause of celiac disease. The engineered enzyme exhibits a k(cat)/K(M) of 568 M(-1) s(-1), representing a 116-fold greater proteolytic activity for a model gluten tetrapeptide than the native template enzyme, as well as an over 800-fold switch in substrate specificity toward immunogenic portions of gluten peptides. The computationally engineered enzyme is resistant to proteolysis by digestive proteases and degrades over 95% of an immunogenic peptide implicated in celiac disease in under an hour. Thus, through identification of a natural enzyme with the pre-existing qualities relevant to an ultimate goal and redefinition of its substrate specificity using computational modeling, we were able to generate an enzyme with potential as a therapeutic for celiac disease.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The ability to rationally modify enzymes to perform novel chemical transformations is essential for the rapid production of next-generation protein therapeutics. Here we describe the use of chemical principles to identify a naturally occurring acid-active peptidase, and the subsequent use of computational protein design tools to reengineer its specificity toward immunogenic elements found in gluten that are the proposed cause of celiac disease. The engineered enzyme exhibits a k(cat)/K(M) of 568 M(-1) s(-1), representing a 116-fold greater proteolytic activity for a model gluten tetrapeptide than the native template enzyme, as well as an over 800-fold switch in substrate specificity toward immunogenic portions of gluten peptides. The computationally engineered enzyme is resistant to proteolysis by digestive proteases and degrades over 95% of an immunogenic peptide implicated in celiac disease in under an hour. Thus, through identification of a natural enzyme with the pre-existing qualities relevant to an ultimate goal and redefinition of its substrate specificity using computational modeling, we were able to generate an enzyme with potential as a therapeutic for celiac disease.
Sarah Baxter, Abigail R Lambert, Ryan Kuhar, Jordan Jarjour, Nadia Kulshina, Fabio Parmeggiani, Patrick Danaher, Jacob Gano, David Baker, Barry L Stoddard, Andrew M Scharenberg
Engineering domain fusion chimeras from I-OnuI family LAGLIDADG homing endonucleases. Journal Article
In: Nucleic acids research, vol. 40, pp. 7985-8000, 2012, ISSN: 1362-4962.
@article{455,
title = {Engineering domain fusion chimeras from I-OnuI family LAGLIDADG homing endonucleases.},
author = { Sarah Baxter and Abigail R Lambert and Ryan Kuhar and Jordan Jarjour and Nadia Kulshina and Fabio Parmeggiani and Patrick Danaher and Jacob Gano and David Baker and Barry L Stoddard and Andrew M Scharenberg},
url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Baxter_NuclAcRes_2012.pdf},
issn = {1362-4962},
year = {2012},
date = {2012-09-01},
journal = {Nucleic acids research},
volume = {40},
pages = {7985-8000},
abstract = {Although engineered LAGLIDADG homing endonucleases (LHEs) are finding increasing applications in biotechnology, their generation remains a challenging, industrial-scale process. As new single-chain LAGLIDADG nuclease scaffolds are identified, however, an alternative paradigm is emerging: identification of an LHE scaffold whose native cleavage site is a close match to a desired target sequence, followed by small-scale engineering to modestly refine recognition specificity. The application of this paradigm could be accelerated if methods were available for fusing N- and C-terminal domains from newly identified LHEs into chimeric enzymes with hybrid cleavage sites. Here we have analyzed the structural requirements for fusion of domains extracted from six single-chain I-OnuI family LHEs, spanning 40-70% amino acid identity. Our analyses demonstrate that both the LAGLIDADG helical interface residues and the linker peptide composition have important effects on the stability and activity of chimeric enzymes. Using a simple domain fusion method in which linker peptide residues predicted to contact their respective domains are retained, and in which limited variation is introduced into the LAGLIDADG helix and nearby interface residues, catalytically active enzymes were recoverable for ~70% of domain chimeras. This method will be useful for creating large numbers of chimeric LHEs for genome engineering applications.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Although engineered LAGLIDADG homing endonucleases (LHEs) are finding increasing applications in biotechnology, their generation remains a challenging, industrial-scale process. As new single-chain LAGLIDADG nuclease scaffolds are identified, however, an alternative paradigm is emerging: identification of an LHE scaffold whose native cleavage site is a close match to a desired target sequence, followed by small-scale engineering to modestly refine recognition specificity. The application of this paradigm could be accelerated if methods were available for fusing N- and C-terminal domains from newly identified LHEs into chimeric enzymes with hybrid cleavage sites. Here we have analyzed the structural requirements for fusion of domains extracted from six single-chain I-OnuI family LHEs, spanning 40-70% amino acid identity. Our analyses demonstrate that both the LAGLIDADG helical interface residues and the linker peptide composition have important effects on the stability and activity of chimeric enzymes. Using a simple domain fusion method in which linker peptide residues predicted to contact their respective domains are retained, and in which limited variation is introduced into the LAGLIDADG helix and nearby interface residues, catalytically active enzymes were recoverable for ~70% of domain chimeras. This method will be useful for creating large numbers of chimeric LHEs for genome engineering applications.
Assaf Alon, Iris Grossman, Yair Gat, Vamsi K Kodali, Frank DiMaio, Tevie Mehlman, Gilad Haran, David Baker, Colin Thorpe, Deborah Fass
The dynamic disulphide relay of quiescin sulphydryl oxidase Journal Article
In: Nature, vol. 488, pp. 414-8, 2012, ISSN: 1476-4687.
@article{600,
title = {The dynamic disulphide relay of quiescin sulphydryl oxidase},
author = { Assaf Alon and Iris Grossman and Yair Gat and Vamsi K Kodali and Frank DiMaio and Tevie Mehlman and Gilad Haran and David Baker and Colin Thorpe and Deborah Fass},
url = {https://www.bakerlab.org/wp-content/uploads/2016/01/thedynamicdisulphide_Baker2012.pdf},
doi = {10.1038/nature11267},
issn = {1476-4687},
year = {2012},
date = {2012-08-01},
journal = {Nature},
volume = {488},
pages = {414-8},
abstract = {Protein stability, assembly, localization and regulation often depend on the formation of disulphide crosslinks between cysteine side chains. Enzymes known as sulphydryl oxidases catalyse de novo disulphide formation and initiate intra- and intermolecular dithiol/disulphide relays to deliver the disulphides to substrate proteins. Quiescin sulphydryl oxidase (QSOX) is a unique, multi-domain disulphide catalyst that is localized primarily to the Golgi apparatus and secreted fluids and has attracted attention owing to its overproduction in tumours. In addition to its physiological importance, QSOX is a mechanistically intriguing enzyme, encompassing functions typically carried out by a series of proteins in other disulphide-formation pathways. How disulphides are relayed through the multiple redox-active sites of QSOX and whether there is a functional benefit to concatenating these sites on a single polypeptide are open questions. Here we present the first crystal structure of an intact QSOX enzyme, derived from a trypanosome parasite. Notably, sequential sites in the disulphide relay were found more than 40 r A apart in this structure, too far for direct disulphide transfer. To resolve this puzzle, we trapped and crystallized an intermediate in the disulphide hand-off, which showed a 165textdegree domain rotation relative to the original structure, bringing the two active sites within disulphide-bonding distance. The comparable structure of a mammalian QSOX enzyme, also presented here, shows further biochemical features that facilitate disulphide transfer in metazoan orthologues. Finally, we quantified the contribution of concatenation to QSOX activity, providing general lessons for the understanding of multi-domain enzymes and the design of new catalytic relays.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Protein stability, assembly, localization and regulation often depend on the formation of disulphide crosslinks between cysteine side chains. Enzymes known as sulphydryl oxidases catalyse de novo disulphide formation and initiate intra- and intermolecular dithiol/disulphide relays to deliver the disulphides to substrate proteins. Quiescin sulphydryl oxidase (QSOX) is a unique, multi-domain disulphide catalyst that is localized primarily to the Golgi apparatus and secreted fluids and has attracted attention owing to its overproduction in tumours. In addition to its physiological importance, QSOX is a mechanistically intriguing enzyme, encompassing functions typically carried out by a series of proteins in other disulphide-formation pathways. How disulphides are relayed through the multiple redox-active sites of QSOX and whether there is a functional benefit to concatenating these sites on a single polypeptide are open questions. Here we present the first crystal structure of an intact QSOX enzyme, derived from a trypanosome parasite. Notably, sequential sites in the disulphide relay were found more than 40 r A apart in this structure, too far for direct disulphide transfer. To resolve this puzzle, we trapped and crystallized an intermediate in the disulphide hand-off, which showed a 165textdegree domain rotation relative to the original structure, bringing the two active sites within disulphide-bonding distance. The comparable structure of a mammalian QSOX enzyme, also presented here, shows further biochemical features that facilitate disulphide transfer in metazoan orthologues. Finally, we quantified the contribution of concatenation to QSOX activity, providing general lessons for the understanding of multi-domain enzymes and the design of new catalytic relays.
2011
Sean J Wu, Christopher B Eiben, John H Carra, Ivan Huang, David Zong, Peixian Liu, Cindy T Wu, Jeff Nivala, Josef Dunbar, Tomas Huber, Jeffrey Senft, Rowena Schokman, Matthew D Smith, Jeremy H Mills, Arthur M Friedlander, David Baker, Justin B Siegel
Improvement of a potential anthrax therapeutic by computational protein design Journal Article
In: The Journal of Biological Chemistry, vol. 286, pp. 32586-92, 2011, ISSN: 1083-351X.
@article{591,
title = {Improvement of a potential anthrax therapeutic by computational protein design},
author = { Sean J Wu and Christopher B Eiben and John H Carra and Ivan Huang and David Zong and Peixian Liu and Cindy T Wu and Jeff Nivala and Josef Dunbar and Tomas Huber and Jeffrey Senft and Rowena Schokman and Matthew D Smith and Jeremy H Mills and Arthur M Friedlander and David Baker and Justin B Siegel},
url = {https://www.bakerlab.org/wp-content/uploads/2018/06/J.-Biol.-Chem.-2011-Wu-32586-92.pdf
http://www.jbc.org/content/286/37/32586},
doi = {10.1074/jbc.M111.251041},
issn = {1083-351X},
year = {2011},
date = {2011-09-01},
journal = {The Journal of Biological Chemistry},
volume = {286},
pages = {32586-92},
abstract = {Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-γ-d-glutamic acid capsule. In the presence of excess CapD, a B. anthracis γ-glutamyl transpeptidase, the protective capsule is degraded, and the immune system can successfully combat infection. Although CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study, we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improved production properties and dramatically increased kinetic thermostability. At 45 textdegreeC, CapD was completely inactive after 5 min, but circularly permuted CapD remained almost entirely active after 30 min. In addition, we identify an amino acid substitution that dramatically decreased transpeptidation activity but not hydrolysis. Subsequently, we show that this mutant had a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum rather than hydrolysis.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-γ-d-glutamic acid capsule. In the presence of excess CapD, a B. anthracis γ-glutamyl transpeptidase, the protective capsule is degraded, and the immune system can successfully combat infection. Although CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study, we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improved production properties and dramatically increased kinetic thermostability. At 45 textdegreeC, CapD was completely inactive after 5 min, but circularly permuted CapD remained almost entirely active after 30 min. In addition, we identify an amino acid substitution that dramatically decreased transpeptidation activity but not hydrolysis. Subsequently, we show that this mutant had a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum rather than hydrolysis.
Stuart A Sievers, John Karanicolas, Howard W Chang, Anni Zhao, Lin Jiang, Onofrio Zirafi, Jason T Stevens, Jan M”unch, David Baker, David Eisenberg
Structure-based design of non-natural amino-acid inhibitors of amyloid fibril formation Journal Article
In: Nature, 2011, ISSN: 1476-4687.
@article{401,
title = {Structure-based design of non-natural amino-acid inhibitors of amyloid fibril formation},
author = { Stuart A Sievers and John Karanicolas and Howard W Chang and Anni Zhao and Lin Jiang and Onofrio Zirafi and Jason T Stevens and Jan M"unch and David Baker and David Eisenberg},
doi = {10.1038/nature10154},
issn = {1476-4687},
year = {2011},
date = {2011-06-01},
journal = {Nature},
abstract = {Many globular and natively disordered proteins can convert into amyloid fibrils. These fibrils are associated with numerous pathologies as well as with normal cellular functions, and frequently form during protein denaturation. Inhibitors of pathological amyloid fibril formation could be useful in the development of therapeutics, provided that the inhibitors were specific enough to avoid interfering with normal processes. Here we show that computer-aided, structure-based design can yield highly specific peptide inhibitors of amyloid formation. Using known atomic structures of segments of amyloid fibrils as templates, we have designed and characterized an all-d-amino-acid inhibitor of the fibril formation of the tau protein associated with Alzheimertextquoterights disease, and a non-natural l-amino-acid inhibitor of an amyloid fibril that enhances sexual transmission of human immunodeficiency virus. Our results indicate that peptides from structure-based designs can disrupt the fibril formation of full-length proteins, including those, such as tau protein, that lack fully ordered native structures. Because the inhibiting peptides have been designed on structures of dual-β-sheet textquoterightsteric zipperstextquoteright, the successful inhibition of amyloid fibril formation strengthens the hypothesis that amyloid spines contain steric zippers.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Many globular and natively disordered proteins can convert into amyloid fibrils. These fibrils are associated with numerous pathologies as well as with normal cellular functions, and frequently form during protein denaturation. Inhibitors of pathological amyloid fibril formation could be useful in the development of therapeutics, provided that the inhibitors were specific enough to avoid interfering with normal processes. Here we show that computer-aided, structure-based design can yield highly specific peptide inhibitors of amyloid formation. Using known atomic structures of segments of amyloid fibrils as templates, we have designed and characterized an all-d-amino-acid inhibitor of the fibril formation of the tau protein associated with Alzheimertextquoterights disease, and a non-natural l-amino-acid inhibitor of an amyloid fibril that enhances sexual transmission of human immunodeficiency virus. Our results indicate that peptides from structure-based designs can disrupt the fibril formation of full-length proteins, including those, such as tau protein, that lack fully ordered native structures. Because the inhibiting peptides have been designed on structures of dual-β-sheet textquoterightsteric zipperstextquoteright, the successful inhibition of amyloid fibril formation strengthens the hypothesis that amyloid spines contain steric zippers.
Javier Guenaga, Pia Dosenovic, Gilad Ofek, David Baker, William R Schief, Peter D Kwong, Gunilla B Karlsson Hedestam, Richard T Wyatt
Heterologous epitope-scaffold prime:boosting immuno-focuses B cell responses to the HIV-1 gp41 2F5 neutralization determinant Journal Article
In: PloS one, vol. 6, pp. e16074, 2011, ISSN: 1932-6203.
@article{357,
title = {Heterologous epitope-scaffold prime:boosting immuno-focuses B cell responses to the HIV-1 gp41 2F5 neutralization determinant},
author = { Javier Guenaga and Pia Dosenovic and Gilad Ofek and David Baker and William R Schief and Peter D Kwong and Gunilla B Karlsson Hedestam and Richard T Wyatt},
issn = {1932-6203},
year = {2011},
date = {2011-00-01},
journal = {PloS one},
volume = {6},
pages = {e16074},
abstract = {The HIV-1 envelope glycoproteins (Env) gp120 and gp41 mediate entry and are the targets for neutralizing antibodies. Within gp41, a continuous epitope defined by the broadly neutralizing antibody 2F5, is one of the few conserved sites accessible to antibodies on the functional HIV Env spike. Recently, as an initial attempt at structure-guided design, we transplanted the 2F5 epitope onto several non-HIV acceptor scaffold proteins that we termed epitope scaffolds (ES). As immunogens, these ES proteins elicited antibodies with exquisite binding specificity matching that of the 2F5 antibody. These novel 2F5 epitope scaffolds presented us with the opportunity to test heterologous prime:boost immunization strategies to selectively boost antibody responses against the engrafted gp41 2F5 epitope. Such strategies might be employed to target conserved but poorly immunogenic sites on the HIV-1 Env, and, more generally, other structurally defined pathogen targets. Here, we assessed ES prime:boosting by measuring epitope specific serum antibody titers by ELISA and B cell responses by ELISpot analysis using both free 2F5 peptide and an unrelated ES protein as probes. We found that the heterologous ES prime:boosting immunization regimen elicits cross-reactive humoral responses to the structurally constrained 2F5 epitope target, and that incorporating a promiscuous T cell helper epitope in the immunogens resulted in higher antibody titers against the 2F5 graft, but did not result in virus neutralization. Interestingly, two epitope scaffolds (ES1 and ES2), which did not elicit a detectable 2F5 epitope-specific response on their own, boosted such responses when primed with the ES5. Together, these results indicate that heterologous ES prime:boost immunization regimens effectively focus the humoral immune response on the structurally defined and immunogen-conserved HIV-1 2F5 epitope.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The HIV-1 envelope glycoproteins (Env) gp120 and gp41 mediate entry and are the targets for neutralizing antibodies. Within gp41, a continuous epitope defined by the broadly neutralizing antibody 2F5, is one of the few conserved sites accessible to antibodies on the functional HIV Env spike. Recently, as an initial attempt at structure-guided design, we transplanted the 2F5 epitope onto several non-HIV acceptor scaffold proteins that we termed epitope scaffolds (ES). As immunogens, these ES proteins elicited antibodies with exquisite binding specificity matching that of the 2F5 antibody. These novel 2F5 epitope scaffolds presented us with the opportunity to test heterologous prime:boost immunization strategies to selectively boost antibody responses against the engrafted gp41 2F5 epitope. Such strategies might be employed to target conserved but poorly immunogenic sites on the HIV-1 Env, and, more generally, other structurally defined pathogen targets. Here, we assessed ES prime:boosting by measuring epitope specific serum antibody titers by ELISA and B cell responses by ELISpot analysis using both free 2F5 peptide and an unrelated ES protein as probes. We found that the heterologous ES prime:boosting immunization regimen elicits cross-reactive humoral responses to the structurally constrained 2F5 epitope target, and that incorporating a promiscuous T cell helper epitope in the immunogens resulted in higher antibody titers against the 2F5 graft, but did not result in virus neutralization. Interestingly, two epitope scaffolds (ES1 and ES2), which did not elicit a detectable 2F5 epitope-specific response on their own, boosted such responses when primed with the ES5. Together, these results indicate that heterologous ES prime:boost immunization regimens effectively focus the humoral immune response on the structurally defined and immunogen-conserved HIV-1 2F5 epitope.
2010
Gilad Ofek, F Javier Guenaga, William R Schief, Jeff Skinner, David Baker, Richard Wyatt, Peter D Kwong
Elicitation of structure-specific antibodies by epitope scaffolds Journal Article
In: Proceedings of the National Academy of Sciences of the United States of America, vol. 107, pp. 17880-7, 2010, ISSN: 1091-6490.
@article{270,
title = {Elicitation of structure-specific antibodies by epitope scaffolds},
author = { Gilad Ofek and F Javier Guenaga and William R Schief and Jeff Skinner and David Baker and Richard Wyatt and Peter D Kwong},
issn = {1091-6490},
year = {2010},
date = {2010-10-01},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {107},
pages = {17880-7},
abstract = {Elicitation of antibodies against targets that are immunorecessive, cryptic, or transient in their native context has been a challenge for vaccine design. Here we demonstrate the elicitation of structure-specific antibodies against the HIV-1 gp41 epitope of the broadly neutralizing antibody 2F5. This conformationally flexible region of gp41 assumes mostly helical conformations but adopts a kinked, extended structure when bound by antibody 2F5. Computational techniques were employed to transplant the 2F5 epitope into select acceptor scaffolds. The resultant "2F5-epitope scaffolds" possessed nanomolar affinity for antibody 2F5 and a range of epitope flexibilities and antigenic specificities. Crystallographic characterization of the epitope scaffold with highest affinity and antigenic discrimination confirmed good to near perfect attainment of the target conformation for the gp41 molecular graft in free and 2F5-bound states, respectively. Animals immunized with 2F5-epitope scaffolds showed levels of graft-specific immune responses that correlated with graft flexibility (p~<~0.04), while antibody responses against the graft-as dissected residue-by-residue with alanine substitutions-resembled more closely those of 2F5 than sera elicited with flexible or cyclized peptides, a resemblance heightened by heterologous prime-boost. Lastly, crystal structures of a gp41 peptide in complex with monoclonal antibodies elicited by the 2F5-epitope scaffolds revealed that the elicited antibodies induce gp41 to assume its 2F5-recognized shape. Epitope scaffolds thus provide a means to elicit antibodies that recognize a predetermined target shape and sequence, even if that shape is transient in nature, and a means by which to dissect factors influencing such elicitation.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Elicitation of antibodies against targets that are immunorecessive, cryptic, or transient in their native context has been a challenge for vaccine design. Here we demonstrate the elicitation of structure-specific antibodies against the HIV-1 gp41 epitope of the broadly neutralizing antibody 2F5. This conformationally flexible region of gp41 assumes mostly helical conformations but adopts a kinked, extended structure when bound by antibody 2F5. Computational techniques were employed to transplant the 2F5 epitope into select acceptor scaffolds. The resultant “2F5-epitope scaffolds” possessed nanomolar affinity for antibody 2F5 and a range of epitope flexibilities and antigenic specificities. Crystallographic characterization of the epitope scaffold with highest affinity and antigenic discrimination confirmed good to near perfect attainment of the target conformation for the gp41 molecular graft in free and 2F5-bound states, respectively. Animals immunized with 2F5-epitope scaffolds showed levels of graft-specific immune responses that correlated with graft flexibility (p~<~0.04), while antibody responses against the graft-as dissected residue-by-residue with alanine substitutions-resembled more closely those of 2F5 than sera elicited with flexible or cyclized peptides, a resemblance heightened by heterologous prime-boost. Lastly, crystal structures of a gp41 peptide in complex with monoclonal antibodies elicited by the 2F5-epitope scaffolds revealed that the elicited antibodies induce gp41 to assume its 2F5-recognized shape. Epitope scaffolds thus provide a means to elicit antibodies that recognize a predetermined target shape and sequence, even if that shape is transient in nature, and a means by which to dissect factors influencing such elicitation.
Bruno E Correia, Yih-En Andrew Ban, Margaret A Holmes, Hengyu Xu, Katharine Ellingson, Zane Kraft, Chris Carrico, Erica Boni, D Noah Sather, Camille Zenobia, Katherine Y Burke, Tyler Bradley-Hewitt, Jessica F Bruhn-Johannsen, Oleksandr Kalyuzhniy, David Baker, Roland K Strong, Leonidas Stamatatos, William R Schief
Computational design of epitope-scaffolds allows induction of antibodies specific for a poorly immunogenic HIV vaccine epitope Journal Article
In: Structure, vol. 18, pp. 1116-26, 2010, ISSN: 1878-4186.
@article{228,
title = {Computational design of epitope-scaffolds allows induction of antibodies specific for a poorly immunogenic HIV vaccine epitope},
author = { Bruno E Correia and Yih-En Andrew Ban and Margaret A Holmes and Hengyu Xu and Katharine Ellingson and Zane Kraft and Chris Carrico and Erica Boni and D Noah Sather and Camille Zenobia and Katherine Y Burke and Tyler Bradley-Hewitt and Jessica F Bruhn-Johannsen and Oleksandr Kalyuzhniy and David Baker and Roland K Strong and Leonidas Stamatatos and William R Schief},
issn = {1878-4186},
year = {2010},
date = {2010-09-01},
journal = {Structure},
volume = {18},
pages = {1116-26},
abstract = {Broadly cross-reactive monoclonal antibodies define epitopes for vaccine development against HIV and other highly mutable viruses. Crystal structures are available for several such antibody-epitope complexes, but methods are needed to translate that structural information into immunogens that re-elicit similar antibodies. We describe a general computational method to design epitope-scaffolds in which contiguous structural epitopes are transplanted to scaffold proteins for conformational stabilization and immune presentation. Epitope-scaffolds designed for the poorly immunogenic but conserved HIV epitope 4E10 exhibited high epitope structural mimicry, bound with higher affinities to monoclonal antibody (mAb) 4E10 than the cognate peptide, and inhibited HIV neutralization by HIV+ sera. Rabbit immunization with an epitope-scaffold induced antibodies with structural specificity highly similar to mAb 4E10, an important advance toward elicitation of neutralizing activity. The results demonstrate that computationally designed epitope-scaffolds are valuable as structure-specific serological reagents and as immunogens to elicit antibodies with predetermined structural specificity.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Broadly cross-reactive monoclonal antibodies define epitopes for vaccine development against HIV and other highly mutable viruses. Crystal structures are available for several such antibody-epitope complexes, but methods are needed to translate that structural information into immunogens that re-elicit similar antibodies. We describe a general computational method to design epitope-scaffolds in which contiguous structural epitopes are transplanted to scaffold proteins for conformational stabilization and immune presentation. Epitope-scaffolds designed for the poorly immunogenic but conserved HIV epitope 4E10 exhibited high epitope structural mimicry, bound with higher affinities to monoclonal antibody (mAb) 4E10 than the cognate peptide, and inhibited HIV neutralization by HIV+ sera. Rabbit immunization with an epitope-scaffold induced antibodies with structural specificity highly similar to mAb 4E10, an important advance toward elicitation of neutralizing activity. The results demonstrate that computationally designed epitope-scaffolds are valuable as structure-specific serological reagents and as immunogens to elicit antibodies with predetermined structural specificity.
Douglas M Fowler, Carlos L Araya, Sarel J Fleishman, Elizabeth H Kellogg, Jason J Stephany, David Baker, Stanley Fields
High-resolution mapping of protein sequence-function relationships Journal Article
In: Nature methods, vol. 7, pp. 741-6, 2010, ISSN: 1548-7105.
@article{267,
title = {High-resolution mapping of protein sequence-function relationships},
author = { Douglas M Fowler and Carlos L Araya and Sarel J Fleishman and Elizabeth H Kellogg and Jason J Stephany and David Baker and Stanley Fields},
issn = {1548-7105},
year = {2010},
date = {2010-09-01},
journal = {Nature methods},
volume = {7},
pages = {741-6},
abstract = {We present a large-scale approach to investigate the functional consequences of sequence variation in a protein. The approach entails the display of hundreds of thousands of protein variants, moderate selection for activity and high-throughput DNA sequencing to quantify the performance of each variant. Using this strategy, we tracked the performance of >600,000 variants of a human WW domain after three and six rounds of selection by phage display for binding to its peptide ligand. Binding properties of these variants defined a high-resolution map of mutational preference across the WW domain; each position had unique features that could not be captured by a few representative mutations. Our approach could be applied to many in vitro or in vivo protein assays, providing a general means for understanding how protein function relates to sequence.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We present a large-scale approach to investigate the functional consequences of sequence variation in a protein. The approach entails the display of hundreds of thousands of protein variants, moderate selection for activity and high-throughput DNA sequencing to quantify the performance of each variant. Using this strategy, we tracked the performance of >600,000 variants of a human WW domain after three and six rounds of selection by phage display for binding to its peptide ligand. Binding properties of these variants defined a high-resolution map of mutational preference across the WW domain; each position had unique features that could not be captured by a few representative mutations. Our approach could be applied to many in vitro or in vivo protein assays, providing a general means for understanding how protein function relates to sequence.
Ben Blum, Michael I Jordan, David Baker
Feature space resampling for protein conformational search Journal Article
In: Proteins, vol. 78, pp. 1583-93, 2010, ISSN: 1097-0134.
@article{271,
title = {Feature space resampling for protein conformational search},
author = { Ben Blum and Michael I Jordan and David Baker},
issn = {1097-0134},
year = {2010},
date = {2010-05-01},
journal = {Proteins},
volume = {78},
pages = {1583-93},
abstract = {De novo protein structure prediction requires location of the lowest energy state of the polypeptide chain among a vast set of possible conformations. Powerful approaches include conformational space annealing, in which search progressively focuses on the most promising regions of conformational space, and genetic algorithms, in which features of the best conformations thus far identified are recombined. We describe a new approach that combines the strengths of these two approaches. Protein conformations are projected onto a discrete feature space which includes backbone torsion angles, secondary structure, and beta pairings. For each of these there is one "native" value: the one found in the native structure. We begin with a large number of conformations generated in independent Monte Carlo structure prediction trajectories from Rosetta. Native values for each feature are predicted from the frequencies of feature value occurrences and the energy distribution in conformations containing them. A second round of structure prediction trajectories are then guided by the predicted native feature distributions. We show that native features can be predicted at much higher than background rates, and that using the predicted feature distributions improves structure prediction in a benchmark of 28 proteins. The advantages of our approach are that features from many different input structures can be combined simultaneously without producing atomic clashes or otherwise physically inviable models, and that the features being recombined have a relatively high chance of being correct.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
De novo protein structure prediction requires location of the lowest energy state of the polypeptide chain among a vast set of possible conformations. Powerful approaches include conformational space annealing, in which search progressively focuses on the most promising regions of conformational space, and genetic algorithms, in which features of the best conformations thus far identified are recombined. We describe a new approach that combines the strengths of these two approaches. Protein conformations are projected onto a discrete feature space which includes backbone torsion angles, secondary structure, and beta pairings. For each of these there is one “native” value: the one found in the native structure. We begin with a large number of conformations generated in independent Monte Carlo structure prediction trajectories from Rosetta. Native values for each feature are predicted from the frequencies of feature value occurrences and the energy distribution in conformations containing them. A second round of structure prediction trajectories are then guided by the predicted native feature distributions. We show that native features can be predicted at much higher than background rates, and that using the predicted feature distributions improves structure prediction in a benchmark of 28 proteins. The advantages of our approach are that features from many different input structures can be combined simultaneously without producing atomic clashes or otherwise physically inviable models, and that the features being recombined have a relatively high chance of being correct.
Charles Chung Yun Leung, Elizabeth Kellogg, Anja Kuhnert, Frank H”anel, David Baker, J N Mark Glover
Insights from the crystal structure of the sixth BRCT domain of topoisomerase IIbeta binding protein 1 Journal Article
In: Protein science : a publication of the Protein Society, vol. 19, pp. 162-7, 2010, ISSN: 1469-896X.
@article{582,
title = {Insights from the crystal structure of the sixth BRCT domain of topoisomerase IIbeta binding protein 1},
author = { Charles Chung Yun Leung and Elizabeth Kellogg and Anja Kuhnert and Frank H"anel and David Baker and J N Mark Glover},
doi = {10.1002/pro.290},
issn = {1469-896X},
year = {2010},
date = {2010-01-01},
journal = {Protein science : a publication of the Protein Society},
volume = {19},
pages = {162-7},
abstract = {Topoisomerase IIbeta binding protein 1 (TopBP1) is a major player in the DNA damage response and interacts with a number of protein partners via its eight BRCA1 carboxy-terminal (BRCT) domains. In particular, the sixth BRCT domain of TopBP1 has been implicated in binding to the phosphorylated transcription factor, E2F1, and poly(ADP-ribose) polymerase 1 (PARP-1), where the latter interaction is responsible for the poly(ADP-ribosyl)ation of TopBP1. To gain a better understanding of the nature of TopBP1 BRCT6 interactions, we solved the crystal structure of BRCT6 to 1.34 A. The crystal structure reveals a degenerate phospho-peptide binding pocket and lacks conserved hydrophobic residues involved in packing of tandem BRCT repeats, which, together with results from phospho-peptide binding studies, strongly suggest that TopBP1 BRCT6 independently does not function as a phospho-peptide binding domain. We further provide insight into poly(ADP-ribose) binding and sites of potential modification by PARP-1.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Topoisomerase IIbeta binding protein 1 (TopBP1) is a major player in the DNA damage response and interacts with a number of protein partners via its eight BRCA1 carboxy-terminal (BRCT) domains. In particular, the sixth BRCT domain of TopBP1 has been implicated in binding to the phosphorylated transcription factor, E2F1, and poly(ADP-ribose) polymerase 1 (PARP-1), where the latter interaction is responsible for the poly(ADP-ribosyl)ation of TopBP1. To gain a better understanding of the nature of TopBP1 BRCT6 interactions, we solved the crystal structure of BRCT6 to 1.34 A. The crystal structure reveals a degenerate phospho-peptide binding pocket and lacks conserved hydrophobic residues involved in packing of tandem BRCT repeats, which, together with results from phospho-peptide binding studies, strongly suggest that TopBP1 BRCT6 independently does not function as a phospho-peptide binding domain. We further provide insight into poly(ADP-ribose) binding and sites of potential modification by PARP-1.
2008
Christine McBeth, Audrey Seamons, Juan C Pizarro, Sarel J Fleishman, David Baker, Tanja Kortemme, Joan M Goverman, Roland K Strong
A new twist in TCR diversity revealed by a forbidden alphabeta TCR Journal Article
In: Journal of molecular biology, vol. 375, pp. 1306-19, 2008, ISSN: 1089-8638.
@article{219,
title = {A new twist in TCR diversity revealed by a forbidden alphabeta TCR},
author = { Christine McBeth and Audrey Seamons and Juan C Pizarro and Sarel J Fleishman and David Baker and Tanja Kortemme and Joan M Goverman and Roland K Strong},
issn = {1089-8638},
year = {2008},
date = {2008-02-01},
journal = {Journal of molecular biology},
volume = {375},
pages = {1306-19},
abstract = {We report crystal structures of a negatively selected T cell receptor (TCR) that recognizes two I-A(u)-restricted myelin basic protein peptides and one of its peptide/major histocompatibility complex (pMHC) ligands. Unusual complementarity-determining region (CDR) structural features revealed by our analyses identify a previously unrecognized mechanism by which the highly variable CDR3 regions define ligand specificity. In addition to the pMHC contact residues contributed by CDR3, the CDR3 residues buried deep within the V alpha/V beta interface exert indirect effects on recognition by influencing the V alpha/V beta interdomain angle. This phenomenon represents an additional mechanism for increasing the potential diversity of the TCR repertoire. Both the direct and indirect effects exerted by CDR residues can impact global TCR/MHC docking. Analysis of the available TCR structures in light of these results highlights the significance of the V alpha/V beta interdomain angle in determining specificity and indicates that TCR/pMHC interface features do not distinguish autoimmune from non-autoimmune class II-restricted TCRs.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We report crystal structures of a negatively selected T cell receptor (TCR) that recognizes two I-A(u)-restricted myelin basic protein peptides and one of its peptide/major histocompatibility complex (pMHC) ligands. Unusual complementarity-determining region (CDR) structural features revealed by our analyses identify a previously unrecognized mechanism by which the highly variable CDR3 regions define ligand specificity. In addition to the pMHC contact residues contributed by CDR3, the CDR3 residues buried deep within the V alpha/V beta interface exert indirect effects on recognition by influencing the V alpha/V beta interdomain angle. This phenomenon represents an additional mechanism for increasing the potential diversity of the TCR repertoire. Both the direct and indirect effects exerted by CDR residues can impact global TCR/MHC docking. Analysis of the available TCR structures in light of these results highlights the significance of the V alpha/V beta interdomain angle in determining specificity and indicates that TCR/pMHC interface features do not distinguish autoimmune from non-autoimmune class II-restricted TCRs.
2007
Ingemar Andr’e, Philip Bradley, Chu Wang, David Baker
Prediction of the structure of symmetrical protein assemblies Journal Article
In: Proceedings of the National Academy of Sciences of the United States of America, vol. 104, pp. 17656-61, 2007, ISSN: 0027-8424.
@article{121,
title = {Prediction of the structure of symmetrical protein assemblies},
author = { Ingemar Andr'e and Philip Bradley and Chu Wang and David Baker},
url = {https://www.bakerlab.org/wp-content/uploads/2016/08/André07A.pdf},
issn = {0027-8424},
year = {2007},
date = {2007-11-01},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {104},
pages = {17656-61},
abstract = {Biological supramolecular systems are commonly built up by the self-assembly of identical protein subunits to produce symmetrical oligomers with cyclical, icosahedral, or helical symmetry that play roles in processes ranging from allosteric control and molecular transport to motor action. The large size of these systems often makes them difficult to structurally characterize using experimental techniques. We have developed a computational protocol to predict the structure of symmetrical protein assemblies based on the structure of a single subunit. The method carries out simultaneous optimization of backbone, side chain, and rigid-body degrees of freedom, while restricting the search space to symmetrical conformations. Using this protocol, we can reconstruct, starting from the structure of a single subunit, the structure of cyclic oligomers and the icosahedral virus capsid of satellite panicum virus using a rigid backbone approximation. We predict the oligomeric state of EscJ from the type III secretion system both in its proposed cyclical and crystallized helical form. Finally, we show that the method can recapitulate the structure of an amyloid-like fibril formed by the peptide NNQQNY from the yeast prion protein Sup35 starting from the amino acid sequence alone and searching the complete space of backbone, side chain, and rigid-body degrees of freedom.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Biological supramolecular systems are commonly built up by the self-assembly of identical protein subunits to produce symmetrical oligomers with cyclical, icosahedral, or helical symmetry that play roles in processes ranging from allosteric control and molecular transport to motor action. The large size of these systems often makes them difficult to structurally characterize using experimental techniques. We have developed a computational protocol to predict the structure of symmetrical protein assemblies based on the structure of a single subunit. The method carries out simultaneous optimization of backbone, side chain, and rigid-body degrees of freedom, while restricting the search space to symmetrical conformations. Using this protocol, we can reconstruct, starting from the structure of a single subunit, the structure of cyclic oligomers and the icosahedral virus capsid of satellite panicum virus using a rigid backbone approximation. We predict the oligomeric state of EscJ from the type III secretion system both in its proposed cyclical and crystallized helical form. Finally, we show that the method can recapitulate the structure of an amyloid-like fibril formed by the peptide NNQQNY from the yeast prion protein Sup35 starting from the amino acid sequence alone and searching the complete space of backbone, side chain, and rigid-body degrees of freedom.
Kiril Tsemekhman, Lukasz Goldschmidt, David Eisenberg, David Baker
Cooperative hydrogen bonding in amyloid formation Journal Article
In: Protein science, vol. 16, pp. 761-4, 2007, ISSN: 0961-8368.
@article{114,
title = {Cooperative hydrogen bonding in amyloid formation},
author = { Kiril Tsemekhman and Lukasz Goldschmidt and David Eisenberg and David Baker},
url = {https://www.bakerlab.org/wp-content/uploads/2016/08/tsemekhman07A.pdf},
issn = {0961-8368},
year = {2007},
date = {2007-04-01},
journal = {Protein science},
volume = {16},
pages = {761-4},
abstract = {Amyloid diseases, including Alzheimertextquoterights and prion diseases, are each associated with unbranched protein fibrils. Each fibril is made of a particular protein, yet they share common properties. One such property is nucleation-dependent fibril growth. Monomers of amyloid-forming proteins can remain in dissolved form for long periods, before rapidly assembly into fibrils. The lag before growth has been attributed to slow kinetics of formation of a nucleus, on which other molecules can deposit to form the fibril. We have explored the energetics of fibril formation, based on the known molecular structure of a fibril-forming peptide from the yeast prion, Sup35, using both classical and quantum (density functional theory) methods. We find that the energetics of fibril formation for the first three layers are cooperative using both methods. This cooperativity is consistent with the observation that formation of amyloid fibrils involves slow nucleation and faster growth.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Amyloid diseases, including Alzheimertextquoterights and prion diseases, are each associated with unbranched protein fibrils. Each fibril is made of a particular protein, yet they share common properties. One such property is nucleation-dependent fibril growth. Monomers of amyloid-forming proteins can remain in dissolved form for long periods, before rapidly assembly into fibrils. The lag before growth has been attributed to slow kinetics of formation of a nucleus, on which other molecules can deposit to form the fibril. We have explored the energetics of fibril formation, based on the known molecular structure of a fibril-forming peptide from the yeast prion, Sup35, using both classical and quantum (density functional theory) methods. We find that the energetics of fibril formation for the first three layers are cooperative using both methods. This cooperativity is consistent with the observation that formation of amyloid fibrils involves slow nucleation and faster growth.
Alexander L Watters, Pritilekha Deka, Colin Corrent, David Callender, Gabriele Varani, Tobin Sosnick, David Baker
The highly cooperative folding of small naturally occurring proteins is likely the result of natural selection Journal Article
In: Cell, vol. 128, pp. 613-24, 2007, ISSN: 0092-8674.
@article{111,
title = {The highly cooperative folding of small naturally occurring proteins is likely the result of natural selection},
author = { Alexander L Watters and Pritilekha Deka and Colin Corrent and David Callender and Gabriele Varani and Tobin Sosnick and David Baker},
issn = {0092-8674},
year = {2007},
date = {2007-02-01},
journal = {Cell},
volume = {128},
pages = {613-24},
abstract = {To illuminate the evolutionary pressure acting on the folding free energy landscapes of naturally occurring proteins, we have systematically characterized the folding free energy landscape of Top7, a computationally designed protein lacking an evolutionary history. Stopped-flow kinetics, circular dichroism, and NMR experiments reveal that there are at least three distinct phases in the folding of Top7, that a nonnative conformation is stable at equilibrium, and that multiple fragments of Top7 are stable in isolation. These results indicate that the folding of Top7 is significantly less cooperative than the folding of similarly sized naturally occurring proteins, suggesting that the cooperative folding and smooth free energy landscapes observed for small naturally occurring proteins are not general properties of polypeptide chains that fold to unique stable structures but are instead a product of natural selection.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
To illuminate the evolutionary pressure acting on the folding free energy landscapes of naturally occurring proteins, we have systematically characterized the folding free energy landscape of Top7, a computationally designed protein lacking an evolutionary history. Stopped-flow kinetics, circular dichroism, and NMR experiments reveal that there are at least three distinct phases in the folding of Top7, that a nonnative conformation is stable at equilibrium, and that multiple fragments of Top7 are stable in isolation. These results indicate that the folding of Top7 is significantly less cooperative than the folding of similarly sized naturally occurring proteins, suggesting that the cooperative folding and smooth free energy landscapes observed for small naturally occurring proteins are not general properties of polypeptide chains that fold to unique stable structures but are instead a product of natural selection.
2006
Amy E Palmer, Marta Giacomello, Tanja Kortemme, S Andrew Hires, Varda Lev-Ram, David Baker, Roger Y Tsien
Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs Journal Article
In: Chemistry & biology, vol. 13, pp. 521-30, 2006, ISSN: 1074-5521.
@article{293,
title = {Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs},
author = { Amy E Palmer and Marta Giacomello and Tanja Kortemme and S Andrew Hires and Varda Lev-Ram and David Baker and Roger Y Tsien},
url = {https://www.bakerlab.org/wp-content/uploads/2016/08/palmer06A.pdf},
issn = {1074-5521},
year = {2006},
date = {2006-05-01},
journal = {Chemistry & biology},
volume = {13},
pages = {521-30},
abstract = {The binding interface of calmodulin and a calmodulin binding peptide were reengineered by computationally designing complementary bumps and holes. This redesign led to the development of sensitive and specific pairs of mutant proteins used to sense Ca(2+) in a second generation of genetically encoded Ca(2+) indicators (cameleons). These cameleons are no longer perturbed by large excesses of native calmodulin, and they display Ca(2+) sensitivities tuned over a 100-fold range (0.6-160 microM). Incorporation of circularly permuted Venus in place of Citrine results in a 3- to 5-fold increase in the dynamic range. These redesigned cameleons show significant improvements over previous versions in the ability to monitor Ca(2+) in the cytoplasm as well as distinct subcellular localizations, such as the plasma membrane of neurons and the mitochondria.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The binding interface of calmodulin and a calmodulin binding peptide were reengineered by computationally designing complementary bumps and holes. This redesign led to the development of sensitive and specific pairs of mutant proteins used to sense Ca(2+) in a second generation of genetically encoded Ca(2+) indicators (cameleons). These cameleons are no longer perturbed by large excesses of native calmodulin, and they display Ca(2+) sensitivities tuned over a 100-fold range (0.6-160 microM). Incorporation of circularly permuted Venus in place of Citrine results in a 3- to 5-fold increase in the dynamic range. These redesigned cameleons show significant improvements over previous versions in the ability to monitor Ca(2+) in the cytoplasm as well as distinct subcellular localizations, such as the plasma membrane of neurons and the mitochondria.
Vanita D Sood, David Baker
Recapitulation and design of protein binding peptide structures and sequences Journal Article
In: Journal of molecular biology, vol. 357, pp. 917-27, 2006, ISSN: 0022-2836.
@article{162,
title = {Recapitulation and design of protein binding peptide structures and sequences},
author = { Vanita D Sood and David Baker},
url = {https://www.bakerlab.org/wp-content/uploads/2016/08/sood06A.pdf},
issn = {0022-2836},
year = {2006},
date = {2006-03-01},
journal = {Journal of molecular biology},
volume = {357},
pages = {917-27},
abstract = {An important objective of computational protein design is the generation of high affinity peptide inhibitors of protein-peptide interactions, both as a precursor to the development of therapeutics aimed at disrupting disease causing complexes, and as a tool to aid investigators in understanding the role of specific complexes in the cell. We have developed a computational approach to increase the affinity of a protein-peptide complex by designing N or C-terminal extensions which interact with the protein outside the canonical peptide binding pocket. In a first in silico test, we show that by simultaneously optimizing the sequence and structure of three to nine residue peptide extensions starting from short (1-6 residue) peptide stubs in the binding pocket of a peptide binding protein, the approach can recover both the conformations and the sequences of known binding peptides. Comparison with phage display and other experimental data suggests that the peptide extension approach recapitulates naturally occurring peptide binding specificity better than fixed backbone design, and that it should be useful for predicting peptide binding specificities from crystal structures. We then experimentally test the approach by designing extensions for p53 and dystroglycan-based peptides predicted to bind with increased affinity to the Mdm2 oncoprotein and to dystrophin, respectively. The measured increases in affinity are modest, revealing some limitations of the method. Based on these in silico and experimental results, we discuss future applications of the approach to the prediction and design of protein-peptide interactions.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
An important objective of computational protein design is the generation of high affinity peptide inhibitors of protein-peptide interactions, both as a precursor to the development of therapeutics aimed at disrupting disease causing complexes, and as a tool to aid investigators in understanding the role of specific complexes in the cell. We have developed a computational approach to increase the affinity of a protein-peptide complex by designing N or C-terminal extensions which interact with the protein outside the canonical peptide binding pocket. In a first in silico test, we show that by simultaneously optimizing the sequence and structure of three to nine residue peptide extensions starting from short (1-6 residue) peptide stubs in the binding pocket of a peptide binding protein, the approach can recover both the conformations and the sequences of known binding peptides. Comparison with phage display and other experimental data suggests that the peptide extension approach recapitulates naturally occurring peptide binding specificity better than fixed backbone design, and that it should be useful for predicting peptide binding specificities from crystal structures. We then experimentally test the approach by designing extensions for p53 and dystroglycan-based peptides predicted to bind with increased affinity to the Mdm2 oncoprotein and to dystrophin, respectively. The measured increases in affinity are modest, revealing some limitations of the method. Based on these in silico and experimental results, we discuss future applications of the approach to the prediction and design of protein-peptide interactions.
Michael J Thompson, Stuart A Sievers, John Karanicolas, Magdalena I Ivanova, David Baker, David Eisenberg
The 3D profile method for identifying fibril-forming segments of proteins Journal Article
In: Proceedings of the National Academy of Sciences of the United States of America, vol. 103, pp. 4074-8, 2006, ISSN: 0027-8424.
@article{163,
title = {The 3D profile method for identifying fibril-forming segments of proteins},
author = { Michael J Thompson and Stuart A Sievers and John Karanicolas and Magdalena I Ivanova and David Baker and David Eisenberg},
url = {https://www.bakerlab.org/wp-content/uploads/2016/08/thompson06A.pdf},
issn = {0027-8424},
year = {2006},
date = {2006-03-01},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {103},
pages = {4074-8},
abstract = {Based on the crystal structure of the cross-beta spine formed by the peptide NNQQNY, we have developed a computational approach for identifying those segments of amyloidogenic proteins that themselves can form amyloid-like fibrils. The approach builds on experiments showing that hexapeptides are sufficient for forming amyloid-like fibrils. Each six-residue peptide of a protein of interest is mapped onto an ensemble of templates, or 3D profile, generated from the crystal structure of the peptide NNQQNY by small displacements of one of the two intermeshed beta-sheets relative to the other. The energy of each mapping of a sequence to the profile is evaluated by using ROSETTADESIGN, and the lowest energy match for a given peptide to the template library is taken as the putative prediction. If the energy of the putative prediction is lower than a threshold value, a prediction of fibril formation is made. This method can reach an accuracy of approximately 80% with a P value of approximately 10(-12) when a conservative energy threshold is used to separate peptides that form fibrils from those that do not. We see enrichment for positive predictions in a set of fibril-forming segments of amyloid proteins, and we illustrate the method with applications to proteins of interest in amyloid research.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Based on the crystal structure of the cross-beta spine formed by the peptide NNQQNY, we have developed a computational approach for identifying those segments of amyloidogenic proteins that themselves can form amyloid-like fibrils. The approach builds on experiments showing that hexapeptides are sufficient for forming amyloid-like fibrils. Each six-residue peptide of a protein of interest is mapped onto an ensemble of templates, or 3D profile, generated from the crystal structure of the peptide NNQQNY by small displacements of one of the two intermeshed beta-sheets relative to the other. The energy of each mapping of a sequence to the profile is evaluated by using ROSETTADESIGN, and the lowest energy match for a given peptide to the template library is taken as the putative prediction. If the energy of the putative prediction is lower than a threshold value, a prediction of fibril formation is made. This method can reach an accuracy of approximately 80% with a P value of approximately 10(-12) when a conservative energy threshold is used to separate peptides that form fibrils from those that do not. We see enrichment for positive predictions in a set of fibril-forming segments of amyloid proteins, and we illustrate the method with applications to proteins of interest in amyloid research.
2004
Carol A Rohl, Charlie E M Strauss, Dylan Chivian, David Baker
Modeling structurally variable regions in homologous proteins with rosetta Journal Article
In: Proteins, vol. 55, pp. 656-77, 2004, ISSN: 1097-0134.
@article{177,
title = {Modeling structurally variable regions in homologous proteins with rosetta},
author = { Carol A Rohl and Charlie E M Strauss and Dylan Chivian and David Baker},
url = {https://www.bakerlab.org/wp-content/uploads/2016/06/rohl04A.pdf},
issn = {1097-0134},
year = {2004},
date = {2004-05-01},
journal = {Proteins},
volume = {55},
pages = {656-77},
abstract = {A major limitation of current comparative modeling methods is the accuracy with which regions that are structurally divergent from homologues of known structure can be modeled. Because structural differences between homologous proteins are responsible for variations in protein function and specificity, the ability to model these differences has important functional consequences. Although existing methods can provide reasonably accurate models of short loop regions, modeling longer structurally divergent regions is an unsolved problem. Here we describe a method based on the de novo structure prediction algorithm, Rosetta, for predicting conformations of structurally divergent regions in comparative models. Initial conformations for short segments are selected from the protein structure database, whereas longer segments are built up by using three- and nine-residue fragments drawn from the database and combined by using the Rosetta algorithm. A gap closure term in the potential in combination with modified Newtontextquoterights method for gradient descent minimization is used to ensure continuity of the peptide backbone. Conformations of variable regions are refined in the context of a fixed template structure using Monte Carlo minimization together with rapid repacking of side-chains to iteratively optimize backbone torsion angles and side-chain rotamers. For short loops, mean accuracies of 0.69, 1.45, and 3.62 A are obtained for 4, 8, and 12 residue loops, respectively. In addition, the method can provide reasonable models of conformations of longer protein segments: predicted conformations of 3A root-mean-square deviation or better were obtained for 5 of 10 examples of segments ranging from 13 to 34 residues. In combination with a sequence alignment algorithm, this method generates complete, ungapped models of protein structures, including regions both similar to and divergent from a homologous structure. This combined method was used to make predictions for 28 protein domains in the Critical Assessment of Protein Structure 4 (CASP 4) and 59 domains in CASP 5, where the method ranked highly among comparative modeling and fold recognition methods. Model accuracy in these blind predictions is dominated by alignment quality, but in the context of accurate alignments, long protein segments can be accurately modeled. Notably, the method correctly predicted the local structure of a 39-residue insertion into a TIM barrel in CASP 5 target T0186.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
A major limitation of current comparative modeling methods is the accuracy with which regions that are structurally divergent from homologues of known structure can be modeled. Because structural differences between homologous proteins are responsible for variations in protein function and specificity, the ability to model these differences has important functional consequences. Although existing methods can provide reasonably accurate models of short loop regions, modeling longer structurally divergent regions is an unsolved problem. Here we describe a method based on the de novo structure prediction algorithm, Rosetta, for predicting conformations of structurally divergent regions in comparative models. Initial conformations for short segments are selected from the protein structure database, whereas longer segments are built up by using three- and nine-residue fragments drawn from the database and combined by using the Rosetta algorithm. A gap closure term in the potential in combination with modified Newtontextquoterights method for gradient descent minimization is used to ensure continuity of the peptide backbone. Conformations of variable regions are refined in the context of a fixed template structure using Monte Carlo minimization together with rapid repacking of side-chains to iteratively optimize backbone torsion angles and side-chain rotamers. For short loops, mean accuracies of 0.69, 1.45, and 3.62 A are obtained for 4, 8, and 12 residue loops, respectively. In addition, the method can provide reasonable models of conformations of longer protein segments: predicted conformations of 3A root-mean-square deviation or better were obtained for 5 of 10 examples of segments ranging from 13 to 34 residues. In combination with a sequence alignment algorithm, this method generates complete, ungapped models of protein structures, including regions both similar to and divergent from a homologous structure. This combined method was used to make predictions for 28 protein domains in the Critical Assessment of Protein Structure 4 (CASP 4) and 59 domains in CASP 5, where the method ranked highly among comparative modeling and fold recognition methods. Model accuracy in these blind predictions is dominated by alignment quality, but in the context of accurate alignments, long protein segments can be accurately modeled. Notably, the method correctly predicted the local structure of a 39-residue insertion into a TIM barrel in CASP 5 target T0186.
Brian Kuhlman, David Baker
Exploring folding free energy landscapes using computational protein design Journal Article
In: Current opinion in structural biology, vol. 14, pp. 89-95, 2004, ISSN: 0959-440X.
@article{574,
title = {Exploring folding free energy landscapes using computational protein design},
author = { Brian Kuhlman and David Baker},
url = {https://www.bakerlab.org/wp-content/uploads/2016/06/exploringfoldingfreeenergy_Baker2004.pdf},
doi = {10.1016/j.sbi.2004.01.002},
issn = {0959-440X},
year = {2004},
date = {2004-02-01},
journal = {Current opinion in structural biology},
volume = {14},
pages = {89-95},
abstract = {Recent advances in computational protein design have allowed exciting new insights into the sequence dependence of protein folding free energy landscapes. Whereas most previous studies have examined the sequence dependence of protein stability and folding kinetics by characterizing naturally occurring proteins and variants of these proteins that contain a small number of mutations, it is now possible to generate and characterize computationally designed proteins that differ significantly from naturally occurring proteins in sequence and/or structure. These computer-generated proteins provide insights into the determinants of protein structure, stability and folding, and make it possible to disentangle the properties of proteins that are the consequence of natural selection from those that reflect the fundamental physical chemistry of polypeptide chains.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Recent advances in computational protein design have allowed exciting new insights into the sequence dependence of protein folding free energy landscapes. Whereas most previous studies have examined the sequence dependence of protein stability and folding kinetics by characterizing naturally occurring proteins and variants of these proteins that contain a small number of mutations, it is now possible to generate and characterize computationally designed proteins that differ significantly from naturally occurring proteins in sequence and/or structure. These computer-generated proteins provide insights into the determinants of protein structure, stability and folding, and make it possible to disentangle the properties of proteins that are the consequence of natural selection from those that reflect the fundamental physical chemistry of polypeptide chains.
2003
William J Wedemeyer, David Baker
Efficient minimization of angle-dependent potentials for polypeptides in internal coordinates Journal Article
In: Proteins, vol. 53, pp. 262-72, 2003, ISSN: 1097-0134.
@article{73,
title = {Efficient minimization of angle-dependent potentials for polypeptides in internal coordinates},
author = { William J Wedemeyer and David Baker},
url = {https://www.bakerlab.org/wp-content/uploads/2016/06/wedemeyer03A.pdf},
issn = {1097-0134},
year = {2003},
date = {2003-11-01},
journal = {Proteins},
volume = {53},
pages = {262-72},
abstract = {Angular potentials play an important role in the refinement of protein structures through angle-dependent restraints (e.g., those determined by cross-correlated relaxations, residual dipolar couplings, and hydrogen bonds). Analytic derivatives of such angular potentials with respect to the dihedral angles of proteins would be useful for optimizing such restraints and other types of angular potentials (i.e., such as we are now introducing into protein structure prediction) but have not been described. In this article, analytic derivatives are calculated for four types of angular potentials and integrated with the efficient recursive derivative calculation methods of Go and coworkers. The formulas are implemented in publicly available software and illustrated by refining a low-resolution protein structure with idealized vector-angle, dipolar-coupling, and hydrogen-bond restraints. The method is now being used routinely to optimize hydrogen-bonding potentials in ROSETTA.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Angular potentials play an important role in the refinement of protein structures through angle-dependent restraints (e.g., those determined by cross-correlated relaxations, residual dipolar couplings, and hydrogen bonds). Analytic derivatives of such angular potentials with respect to the dihedral angles of proteins would be useful for optimizing such restraints and other types of angular potentials (i.e., such as we are now introducing into protein structure prediction) but have not been described. In this article, analytic derivatives are calculated for four types of angular potentials and integrated with the efficient recursive derivative calculation methods of Go and coworkers. The formulas are implemented in publicly available software and illustrated by refining a low-resolution protein structure with idealized vector-angle, dipolar-coupling, and hydrogen-bond restraints. The method is now being used routinely to optimize hydrogen-bonding potentials in ROSETTA.
Ruslan I Sadreyev, David Baker, Nick V Grishin
Profile-profile comparisons by COMPASS predict intricate homologies between protein families Journal Article
In: Protein science, vol. 12, pp. 2262-72, 2003, ISSN: 0961-8368.
@article{316,
title = {Profile-profile comparisons by COMPASS predict intricate homologies between protein families},
author = { Ruslan I Sadreyev and David Baker and Nick V Grishin},
url = {https://www.bakerlab.org/wp-content/uploads/2016/06/sadreyev03A.pdf},
issn = {0961-8368},
year = {2003},
date = {2003-10-01},
journal = {Protein science},
volume = {12},
pages = {2262-72},
abstract = {Recently we proposed a novel method of alignment-alignment comparison, COMPASS (the tool for COmparison of Multiple Protein Alignments with Assessment of Statistical Significance). Here we present several examples of the relations between PFAM protein families that were detected by COMPASS and that lead to the predictions of presently unresolved protein structures. We discuss relatively straightforward COMPASS predictions that are new and interesting to us, and that would require a substantial time and effort to justify even for a skilled PSI-BLAST user. All of the presented COMPASS hits are independently confirmed by other methods, including the ab initio structure-prediction method ROSETTA. The tertiary structure predictions made by ROSETTA proved to be useful for improving sequence-derived alignments, because they are based on a reasonable folding of the polypeptide chain rather than on the information from sequence databases. The ability of COMPASS to predict new relations within the PFAM database indicates the high sensitivity of COMPASS searches and substantiates its potential value for the discovery of previously unknown similarities between protein families.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Recently we proposed a novel method of alignment-alignment comparison, COMPASS (the tool for COmparison of Multiple Protein Alignments with Assessment of Statistical Significance). Here we present several examples of the relations between PFAM protein families that were detected by COMPASS and that lead to the predictions of presently unresolved protein structures. We discuss relatively straightforward COMPASS predictions that are new and interesting to us, and that would require a substantial time and effort to justify even for a skilled PSI-BLAST user. All of the presented COMPASS hits are independently confirmed by other methods, including the ab initio structure-prediction method ROSETTA. The tertiary structure predictions made by ROSETTA proved to be useful for improving sequence-derived alignments, because they are based on a reasonable folding of the polypeptide chain rather than on the information from sequence databases. The ability of COMPASS to predict new relations within the PFAM database indicates the high sensitivity of COMPASS searches and substantiates its potential value for the discovery of previously unknown similarities between protein families.
Dmitry N Ivankov, Sergiy O Garbuzynskiy, Eric Alm, Kevin W Plaxco, David Baker, Alexei V Finkelstein
Contact order revisited: influence of protein size on the folding rate Journal Article
In: Protein science, vol. 12, pp. 2057-62, 2003, ISSN: 0961-8368.
@article{84,
title = {Contact order revisited: influence of protein size on the folding rate},
author = { Dmitry N Ivankov and Sergiy O Garbuzynskiy and Eric Alm and Kevin W Plaxco and David Baker and Alexei V Finkelstein},
url = {https://www.bakerlab.org/wp-content/uploads/2016/06/ivankov03A.pdf},
issn = {0961-8368},
year = {2003},
date = {2003-09-01},
journal = {Protein science},
volume = {12},
pages = {2057-62},
abstract = {Guided by the recent success of empirical model predicting the folding rates of small two-state folding proteins from the relative contact order (CO) of their native structures, by a theoretical model of protein folding that predicts that logarithm of the folding rate decreases with the protein chain length L as L(2/3), and by the finding that the folding rates of multistate folding proteins strongly correlate with their sizes and have very bad correlation with CO, we reexamined the dependence of folding rate on CO and L in attempt to find a structural parameter that determines folding rates for the totality of proteins. We show that the Abs_CO = CO x L, is able to predict rather accurately folding rates for both two-state and multistate folding proteins, as well as short peptides, and that this Abs_CO scales with the protein chain length as L(0.70 +/- 0.07) for the totality of studied single-domain proteins and peptides.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Guided by the recent success of empirical model predicting the folding rates of small two-state folding proteins from the relative contact order (CO) of their native structures, by a theoretical model of protein folding that predicts that logarithm of the folding rate decreases with the protein chain length L as L(2/3), and by the finding that the folding rates of multistate folding proteins strongly correlate with their sizes and have very bad correlation with CO, we reexamined the dependence of folding rate on CO and L in attempt to find a structural parameter that determines folding rates for the totality of proteins. We show that the Abs_CO = CO x L, is able to predict rather accurately folding rates for both two-state and multistate folding proteins, as well as short peptides, and that this Abs_CO scales with the protein chain length as L(0.70 +/- 0.07) for the totality of studied single-domain proteins and peptides.
Michelle Scalley-Kim, Philippe Minard, David Baker
Low free energy cost of very long loop insertions in proteins Journal Article
In: Protein science, vol. 12, pp. 197-206, 2003, ISSN: 0961-8368.
@article{76,
title = {Low free energy cost of very long loop insertions in proteins},
author = { Michelle Scalley-Kim and Philippe Minard and David Baker},
url = {https://www.bakerlab.org/wp-content/uploads/2016/06/scalley-kim03A.pdf},
issn = {0961-8368},
year = {2003},
date = {2003-02-01},
journal = {Protein science},
volume = {12},
pages = {197-206},
abstract = {Long insertions into a loop of a folded host protein are expected to have destabilizing effects because of the entropic cost associated with loop closure unless the inserted sequence adopts a folded structure with amino- and carboxy-termini in close proximity. A loop entropy reduction screen based on this concept was used in an attempt to retrieve folded sequences from random sequence libraries. A library of long random sequences was inserted into a loop of the SH2 domain, displayed on the surface of M13 phage, and the inserted sequences that did not disrupt SH2 function were retrieved by panning using beads coated with a phosphotyrosine containing SH2 peptide ligand. Two sequences of a library of 2 x 10(8) sequences were isolated after multiple rounds of panning, and were found to have recovery levels similar to the wild-type SH2 domain and to be relatively intolerant to further mutation in PCR mutagenesis experiments. Surprisingly, although these inserted sequences exhibited little nonrandom structure, they do not significantly destabilize the host SH2 domain. Additional insertion variants recovered at lower levels in the panning experiments were also found to have a minimal effect on the stability and peptide-binding function of the SH2 domain. The additional level of selection present in the panning experiments is likely to involve in vivo folding and assembly, as there was a rough correlation between recovery levels in the phage-panning experiments and protein solubility. The finding that loop insertions of 60-80 amino acids have minimal effects on SH2 domain stability suggests that the free energy cost of inserting long loops may be considerably less than polymer theory estimates based on the entropic cost of loop closure, and, hence, that loop insertion may have provided an evolutionary route to multidomain protein structures.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Long insertions into a loop of a folded host protein are expected to have destabilizing effects because of the entropic cost associated with loop closure unless the inserted sequence adopts a folded structure with amino- and carboxy-termini in close proximity. A loop entropy reduction screen based on this concept was used in an attempt to retrieve folded sequences from random sequence libraries. A library of long random sequences was inserted into a loop of the SH2 domain, displayed on the surface of M13 phage, and the inserted sequences that did not disrupt SH2 function were retrieved by panning using beads coated with a phosphotyrosine containing SH2 peptide ligand. Two sequences of a library of 2 x 10(8) sequences were isolated after multiple rounds of panning, and were found to have recovery levels similar to the wild-type SH2 domain and to be relatively intolerant to further mutation in PCR mutagenesis experiments. Surprisingly, although these inserted sequences exhibited little nonrandom structure, they do not significantly destabilize the host SH2 domain. Additional insertion variants recovered at lower levels in the panning experiments were also found to have a minimal effect on the stability and peptide-binding function of the SH2 domain. The additional level of selection present in the panning experiments is likely to involve in vivo folding and assembly, as there was a rough correlation between recovery levels in the phage-panning experiments and protein solubility. The finding that loop insertions of 60-80 amino acids have minimal effects on SH2 domain stability suggests that the free energy cost of inserting long loops may be considerably less than polymer theory estimates based on the entropic cost of loop closure, and, hence, that loop insertion may have provided an evolutionary route to multidomain protein structures.
2001
V Grantcharova, E J Alm, David Baker, A L Horwich
Mechanisms of protein folding. Journal Article
In: Current opinion in structural biology, vol. 11, pp. 70-82, 2001, ISSN: 0959-440X.
@article{66,
title = {Mechanisms of protein folding.},
author = { V Grantcharova and E J Alm and David Baker and A L Horwich},
issn = {0959-440X},
year = {2001},
date = {2001-02-01},
journal = {Current opinion in structural biology},
volume = {11},
pages = {70-82},
abstract = {The strong correlation between protein folding rates and the contact order suggests that folding rates are largely determined by the topology of the native structure. However, for a given topology, there may be several possible low free energy paths to the native state and the path that is chosen (the lowest free energy path) may depend on differences in interaction energies and local free energies of ordering in different parts of the structure. For larger proteins whose folding is assisted by chaperones, such as the Escherichia coli chaperonin GroEL, advances have been made in understanding both the aspects of an unfolded protein that GroEL recognizes and the mode of binding to the chaperonin. The possibility that GroEL can remove non-native proteins from kinetic traps by unfolding them either during polypeptide binding to the chaperonin or during the subsequent ATP-dependent formation of folding-active complexes with the co-chaperonin GroES has also been explored.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The strong correlation between protein folding rates and the contact order suggests that folding rates are largely determined by the topology of the native structure. However, for a given topology, there may be several possible low free energy paths to the native state and the path that is chosen (the lowest free energy path) may depend on differences in interaction energies and local free energies of ordering in different parts of the structure. For larger proteins whose folding is assisted by chaperones, such as the Escherichia coli chaperonin GroEL, advances have been made in understanding both the aspects of an unfolded protein that GroEL recognizes and the mode of binding to the chaperonin. The possibility that GroEL can remove non-native proteins from kinetic traps by unfolding them either during polypeptide binding to the chaperonin or during the subsequent ATP-dependent formation of folding-active complexes with the co-chaperonin GroES has also been explored.
2000
P M Bowers, C E Strauss, David Baker
De novo protein structure determination using sparse NMR data. Journal Article
In: Journal of biomolecular NMR, vol. 18, pp. 311-8, 2000, ISSN: 0925-2738.
@article{193,
title = {De novo protein structure determination using sparse NMR data.},
author = { P M Bowers and C E Strauss and David Baker},
issn = {0925-2738},
year = {2000},
date = {2000-12-01},
journal = {Journal of biomolecular NMR},
volume = {18},
pages = {311-8},
abstract = {We describe a method for generating moderate to high-resolution protein structures using limited NMR data combined with the ab initio protein structure prediction method Rosetta. Peptide fragments are selected from proteins of known structure based on sequence similarity and consistency with chemical shift and NOE data. Models are built from these fragments by minimizing an energy function that favors hydrophobic burial, strand pairing, and satisfaction of NOE constraints. Models generated using this procedure with approximately 1 NOE constraint per residue are in some cases closer to the corresponding X-ray structures than the published NMR solution structures. The method requires only the sparse constraints available during initial stages of NMR structure determination, and thus holds promise for increasing the speed with which protein solution structures can be determined.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We describe a method for generating moderate to high-resolution protein structures using limited NMR data combined with the ab initio protein structure prediction method Rosetta. Peptide fragments are selected from proteins of known structure based on sequence similarity and consistency with chemical shift and NOE data. Models are built from these fragments by minimizing an energy function that favors hydrophobic burial, strand pairing, and satisfaction of NOE constraints. Models generated using this procedure with approximately 1 NOE constraint per residue are in some cases closer to the corresponding X-ray structures than the published NMR solution structures. The method requires only the sparse constraints available during initial stages of NMR structure determination, and thus holds promise for increasing the speed with which protein solution structures can be determined.
C Bystroff, V Thorsson, David Baker
HMMSTR: a hidden Markov model for local sequence-structure correlations in proteins Journal Article
In: Journal of molecular biology, vol. 301, pp. 173-90, 2000, ISSN: 0022-2836.
@article{194,
title = {HMMSTR: a hidden Markov model for local sequence-structure correlations in proteins},
author = { C Bystroff and V Thorsson and David Baker},
url = {https://www.bakerlab.org/wp-content/uploads/2016/06/bystroff00A.pdf},
issn = {0022-2836},
year = {2000},
date = {2000-08-01},
journal = {Journal of molecular biology},
volume = {301},
pages = {173-90},
abstract = {We describe a hidden Markov model, HMMSTR, for general protein sequence based on the I-sites library of sequence-structure motifs. Unlike the linear hidden Markov models used to model individual protein families, HMMSTR has a highly branched topology and captures recurrent local features of protein sequences and structures that transcend protein family boundaries. The model extends the I-sites library by describing the adjacencies of different sequence-structure motifs as observed in the protein database and, by representing overlapping motifs in a much more compact form, achieves a great reduction in parameters. The HMM attributes a considerably higher probability to coding sequence than does an equivalent dipeptide model, predicts secondary structure with an accuracy of 74.3 %, backbone torsion angles better than any previously reported method and the structural context of beta strands and turns with an accuracy that should be useful for tertiary structure prediction.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We describe a hidden Markov model, HMMSTR, for general protein sequence based on the I-sites library of sequence-structure motifs. Unlike the linear hidden Markov models used to model individual protein families, HMMSTR has a highly branched topology and captures recurrent local features of protein sequences and structures that transcend protein family boundaries. The model extends the I-sites library by describing the adjacencies of different sequence-structure motifs as observed in the protein database and, by representing overlapping motifs in a much more compact form, achieves a great reduction in parameters. The HMM attributes a considerably higher probability to coding sequence than does an equivalent dipeptide model, predicts secondary structure with an accuracy of 74.3 %, backbone torsion angles better than any previously reported method and the structural context of beta strands and turns with an accuracy that should be useful for tertiary structure prediction.
David Baker
A surprising simplicity to protein folding Journal Article
In: Nature, vol. 405, pp. 39-42, 2000, ISSN: 0028-0836.
@article{192,
title = {A surprising simplicity to protein folding},
author = { David Baker},
url = {https://www.bakerlab.org/wp-content/uploads/2016/06/baker00A.pdf},
issn = {0028-0836},
year = {2000},
date = {2000-05-01},
journal = {Nature},
volume = {405},
pages = {39-42},
abstract = {The polypeptide chains that make up proteins have thousands of atoms and hence millions of possible inter-atomic interactions. It might be supposed that the resulting complexity would make prediction of protein structure and protein-folding mechanisms nearly impossible. But the fundamental physics underlying folding may be much simpler than this complexity would lead us to expect folding rates and mechanisms appear to be largely determined by the topology of the native (folded) state, and new methods have shown great promise in predicting protein-folding mechanisms and the three-dimensional structures of proteins.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The polypeptide chains that make up proteins have thousands of atoms and hence millions of possible inter-atomic interactions. It might be supposed that the resulting complexity would make prediction of protein structure and protein-folding mechanisms nearly impossible. But the fundamental physics underlying folding may be much simpler than this complexity would lead us to expect folding rates and mechanisms appear to be largely determined by the topology of the native (folded) state, and new methods have shown great promise in predicting protein-folding mechanisms and the three-dimensional structures of proteins.
1999
M L Scalley, S Nauli, S T Gladwin, David Baker
Structural transitions in the protein L denatured state ensemble Journal Article
In: Biochemistry, vol. 38, pp. 15927-35, 1999, ISSN: 0006-2960.
@article{42,
title = {Structural transitions in the protein L denatured state ensemble},
author = { M L Scalley and S Nauli and S T Gladwin and David Baker},
url = {https://www.bakerlab.org/wp-content/uploads/2016/06/scalley99A.pdf},
issn = {0006-2960},
year = {1999},
date = {1999-11-01},
journal = {Biochemistry},
volume = {38},
pages = {15927-35},
abstract = {We use a broad array of biophysical methods to probe the extent of structure and time scale of structural transitions in the protein L denatured state ensemble. Measurement of amide proton exchange protection during the first several milliseconds following initiation of refolding in 0.4 M sodium sulfate revealed weak protection in the first beta-hairpin and helix. A tryptophan residue was introduced into the first beta-hairpin to probe the extent of structure formation in this part of the protein; the intrinsic fluorescence of this tryptophan was found to deviate from that expected given its local sequence context in 2-3 M guanidine, suggesting some partial ordering of this region in the unfolded state ensemble. To further probe this partial ordering, dansyl groups were introduced via cysteine residues at three sites in the protein. It was found that fluorescence energy transfer from the introduced tryptophan to the dansyl groups decreased dramatically upon unfolding. Stopped-flow fluorescence studies showed that the recovery of dansyl fluorescence upon refolding occurred on a submillisecond time scale. To probe the interactions responsible for the residual structure observed in the denatured state ensemble, the conformation of a peptide corresponding to the first beta-hairpin and helix of protein L was studied using circular dichroism spectroscopy and compared to that of full-length protein L and previously characterized peptides corresponding to the isolated helix and second beta-hairpin.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We use a broad array of biophysical methods to probe the extent of structure and time scale of structural transitions in the protein L denatured state ensemble. Measurement of amide proton exchange protection during the first several milliseconds following initiation of refolding in 0.4 M sodium sulfate revealed weak protection in the first beta-hairpin and helix. A tryptophan residue was introduced into the first beta-hairpin to probe the extent of structure formation in this part of the protein; the intrinsic fluorescence of this tryptophan was found to deviate from that expected given its local sequence context in 2-3 M guanidine, suggesting some partial ordering of this region in the unfolded state ensemble. To further probe this partial ordering, dansyl groups were introduced via cysteine residues at three sites in the protein. It was found that fluorescence energy transfer from the introduced tryptophan to the dansyl groups decreased dramatically upon unfolding. Stopped-flow fluorescence studies showed that the recovery of dansyl fluorescence upon refolding occurred on a submillisecond time scale. To probe the interactions responsible for the residual structure observed in the denatured state ensemble, the conformation of a peptide corresponding to the first beta-hairpin and helix of protein L was studied using circular dichroism spectroscopy and compared to that of full-length protein L and previously characterized peptides corresponding to the isolated helix and second beta-hairpin.
1998
V P Grantcharova, D S Riddle, J V Santiago, David Baker
Important role of hydrogen bonds in the structurally polarized transition state for folding of the src SH3 domain Journal Article
In: Nature structural biology, vol. 5, pp. 714-20, 1998, ISSN: 1072-8368.
@article{204,
title = {Important role of hydrogen bonds in the structurally polarized transition state for folding of the src SH3 domain},
author = { V P Grantcharova and D S Riddle and J V Santiago and David Baker},
url = {https://www.bakerlab.org/wp-content/uploads/2016/06/grantcharova98A.pdf},
issn = {1072-8368},
year = {1998},
date = {1998-08-01},
journal = {Nature structural biology},
volume = {5},
pages = {714-20},
abstract = {Experimental and theoretical studies on the folding of small proteins such as the chymotrypsin inhibitor 2 (CI-2) and the P22 Arc repressor suggest that the folding transition state is an expanded version of the native state with most interactions partially formed. Here we report that this picture does not hold generally: a hydrogen bond network involving two beta-turns and an adjacent hydrophobic cluster appear to be formed in the folding transition state of the src SH3 domain, while the remainder of the polypeptide chain is largely unstructured. Comparison with data on other small proteins suggests that this structural polarization is a consequence of the topology of the SH3 domain fold. The non-uniform distribution of structure in the folding transition state provides a challenging test for computational models of the folding process.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Experimental and theoretical studies on the folding of small proteins such as the chymotrypsin inhibitor 2 (CI-2) and the P22 Arc repressor suggest that the folding transition state is an expanded version of the native state with most interactions partially formed. Here we report that this picture does not hold generally: a hydrogen bond network involving two beta-turns and an adjacent hydrophobic cluster appear to be formed in the folding transition state of the src SH3 domain, while the remainder of the polypeptide chain is largely unstructured. Comparison with data on other small proteins suggests that this structural polarization is a consequence of the topology of the SH3 domain fold. The non-uniform distribution of structure in the folding transition state provides a challenging test for computational models of the folding process.
D E Kim, H Gu, D Baker
The sequences of small proteins are not extensively optimized for rapid folding by natural selection Journal Article
In: Proceedings of the National Academy of Sciences of the United States of America, vol. 95, pp. 4982-6, 1998, ISSN: 0027-8424.
@article{312,
title = {The sequences of small proteins are not extensively optimized for rapid folding by natural selection},
author = { D E Kim and H Gu and D Baker},
url = {https://www.bakerlab.org/wp-content/uploads/2016/06/kim98A.pdf},
issn = {0027-8424},
year = {1998},
date = {1998-04-01},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {95},
pages = {4982-6},
abstract = {The thermodynamic stabilities of small protein domains are clearly subject to natural selection, but it is less clear whether the rapid folding rates typically observed for such proteins are consequences of direct evolutionary optimization or reflect intrinsic physical properties of the polypeptide chain. This issue can be investigated by comparing the folding rates of laboratory-generated protein sequences to those of naturally occurring sequences provided that the method by which the sequences are generated has no kinetic bias. Herein we report the folding thermodynamics and kinetics of 12 heavily mutated variants of the small IgG binding domain of protein L retrieved from high-complexity combinatorial libraries by using a phage-display selection for proper folding that does not discriminate between rapidly and slowly folding proteins. Although the stabilities of all variants were decreased, many of the variants fold faster than wild type. Taken together with similar results for the src homology 3 domain, this observation suggests that the sequences of small proteins have not been extensively optimized for rapid folding; instead, rapid folding appears to be a consequence of selection for stability.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The thermodynamic stabilities of small protein domains are clearly subject to natural selection, but it is less clear whether the rapid folding rates typically observed for such proteins are consequences of direct evolutionary optimization or reflect intrinsic physical properties of the polypeptide chain. This issue can be investigated by comparing the folding rates of laboratory-generated protein sequences to those of naturally occurring sequences provided that the method by which the sequences are generated has no kinetic bias. Herein we report the folding thermodynamics and kinetics of 12 heavily mutated variants of the small IgG binding domain of protein L retrieved from high-complexity combinatorial libraries by using a phage-display selection for proper folding that does not discriminate between rapidly and slowly folding proteins. Although the stabilities of all variants were decreased, many of the variants fold faster than wild type. Taken together with similar results for the src homology 3 domain, this observation suggests that the sequences of small proteins have not been extensively optimized for rapid folding; instead, rapid folding appears to be a consequence of selection for stability.
K W Plaxco, K T Simons, David Baker
Contact order, transition state placement and the refolding rates of single domain proteins Journal Article
In: Journal of molecular biology, vol. 277, pp. 985-94, 1998, ISSN: 0022-2836.
@article{207,
title = {Contact order, transition state placement and the refolding rates of single domain proteins},
author = { K W Plaxco and K T Simons and David Baker},
url = {https://www.bakerlab.org/wp-content/uploads/2016/06/plaxco98B.pdf},
issn = {0022-2836},
year = {1998},
date = {1998-04-01},
journal = {Journal of molecular biology},
volume = {277},
pages = {985-94},
abstract = {Theoretical studies have suggested relationships between the size, stability and topology of a protein fold and the rate and mechanisms by which it is achieved. The recent characterization of the refolding of a number of simple, single domain proteins has provided a means of testing these assertions. Our investigations have revealed statistically significant correlations between the average sequence separation between contacting residues in the native state and the rate and transition state placement of folding for a non-homologous set of simple, single domain proteins. These indicate that proteins featuring primarily sequence-local contacts tend to fold more rapidly and exhibit less compact folding transition states than those characterized by more non-local interactions. No significant relationship is apparent between protein length and folding rates, but a weak correlation is observed between length and the fraction of solvent-exposed surface area buried in the transition state. Anticipated strong relationships between equilibrium folding free energy and folding kinetics, or between chemical denaturant and temperature dependence-derived measures of transition state placement, are not apparent. The observed correlations are consistent with a model of protein folding in which the size and stability of the polypeptide segments organized in the transition state are largely independent of protein length, but are related to the topological complexity of the native state. The correlation between topological complexity and folding rates may reflect chain entropy contributions to the folding barrier.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Theoretical studies have suggested relationships between the size, stability and topology of a protein fold and the rate and mechanisms by which it is achieved. The recent characterization of the refolding of a number of simple, single domain proteins has provided a means of testing these assertions. Our investigations have revealed statistically significant correlations between the average sequence separation between contacting residues in the native state and the rate and transition state placement of folding for a non-homologous set of simple, single domain proteins. These indicate that proteins featuring primarily sequence-local contacts tend to fold more rapidly and exhibit less compact folding transition states than those characterized by more non-local interactions. No significant relationship is apparent between protein length and folding rates, but a weak correlation is observed between length and the fraction of solvent-exposed surface area buried in the transition state. Anticipated strong relationships between equilibrium folding free energy and folding kinetics, or between chemical denaturant and temperature dependence-derived measures of transition state placement, are not apparent. The observed correlations are consistent with a model of protein folding in which the size and stability of the polypeptide segments organized in the transition state are largely independent of protein length, but are related to the topological complexity of the native state. The correlation between topological complexity and folding rates may reflect chain entropy contributions to the folding barrier.
Q Yi, C Bystroff, P Rajagopal, R E Klevit, David Baker
Prediction and structural characterization of an independently folding substructure in the src SH3 domain Journal Article
In: Journal of molecular biology, vol. 283, pp. 293-300, 1998, ISSN: 0022-2836.
@article{212,
title = {Prediction and structural characterization of an independently folding substructure in the src SH3 domain},
author = { Q Yi and C Bystroff and P Rajagopal and R E Klevit and David Baker},
url = {https://www.bakerlab.org/wp-content/uploads/2016/06/yi98A.pdf},
issn = {0022-2836},
year = {1998},
date = {1998-00-01},
journal = {Journal of molecular biology},
volume = {283},
pages = {293-300},
abstract = {Previous studies of the conformations of peptides spanning the length of the alpha-spectrin SH3 domain suggested that SH3 domains lack independently folding substructures. Using a local structure prediction method based on the I-sites library of sequence-structure motifs, we identified a seven residue peptide in the src SH3 domain predicted to adopt a native-like structure, a type II beta-turn bridging unpaired beta-strands, that was not contained intact in any of the SH3 domain peptides studied earlier. NMR characterization confirmed that the isolated peptide, FKKGERL, adopts a structure similar to that adopted in the native protein: the NOE and 3JNHalpha coupling constant patterns were indicative of a type II beta-turn, and NOEs between the Phe and the Leu side-chains suggest that they are juxtaposed as in the prediction and the native structure. These results support the idea that high-confidence I-sites predictions identify protein segments that are likely to form native-like structures early in folding.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Previous studies of the conformations of peptides spanning the length of the alpha-spectrin SH3 domain suggested that SH3 domains lack independently folding substructures. Using a local structure prediction method based on the I-sites library of sequence-structure motifs, we identified a seven residue peptide in the src SH3 domain predicted to adopt a native-like structure, a type II beta-turn bridging unpaired beta-strands, that was not contained intact in any of the SH3 domain peptides studied earlier. NMR characterization confirmed that the isolated peptide, FKKGERL, adopts a structure similar to that adopted in the native protein: the NOE and 3JNHalpha coupling constant patterns were indicative of a type II beta-turn, and NOEs between the Phe and the Leu side-chains suggest that they are juxtaposed as in the prediction and the native structure. These results support the idea that high-confidence I-sites predictions identify protein segments that are likely to form native-like structures early in folding.
1997
D S Riddle, J V Santiago, S T Bray-Hall, N Doshi, V P Grantcharova, Q Yi, David Baker
Functional rapidly folding proteins from simplified amino acid sequences Journal Article
In: Nature structural biology, vol. 4, pp. 805-9, 1997, ISSN: 1072-8368.
@article{26,
title = {Functional rapidly folding proteins from simplified amino acid sequences},
author = { D S Riddle and J V Santiago and S T Bray-Hall and N Doshi and V P Grantcharova and Q Yi and David Baker},
issn = {1072-8368},
year = {1997},
date = {1997-10-01},
journal = {Nature structural biology},
volume = {4},
pages = {805-9},
abstract = {Early protein synthesis is thought to have involved a reduced amino acid alphabet. What is the minimum number of amino acids that would have been needed to encode complex protein folds similar to those found in nature today? Here we show that a small beta-sheet protein, the SH3 domain, can be largely encoded by a five letter amino acid alphabet but not by a three letter alphabet. Furthermore, despite the dramatic changes in sequence, the folding rates of the reduced alphabet proteins are very close to that of the naturally occurring SH3 domain. This finding suggests that despite the vast size of the search space, the rapid folding of biological sequences to their native states is not the result of extensive evolutionary optimization. Instead, the results support the idea that the interactions which stabilize the native state induce a funnel shape to the free energy landscape sufficient to guide the folding polypeptide chain to the proper structure.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Early protein synthesis is thought to have involved a reduced amino acid alphabet. What is the minimum number of amino acids that would have been needed to encode complex protein folds similar to those found in nature today? Here we show that a small beta-sheet protein, the SH3 domain, can be largely encoded by a five letter amino acid alphabet but not by a three letter alphabet. Furthermore, despite the dramatic changes in sequence, the folding rates of the reduced alphabet proteins are very close to that of the naturally occurring SH3 domain. This finding suggests that despite the vast size of the search space, the rapid folding of biological sequences to their native states is not the result of extensive evolutionary optimization. Instead, the results support the idea that the interactions which stabilize the native state induce a funnel shape to the free energy landscape sufficient to guide the folding polypeptide chain to the proper structure.
1996
C Bystroff, K T Simons, K F Han, David Baker
Local sequence-structure correlations in proteins Journal Article
In: Current opinion in biotechnology, vol. 7, pp. 417-21, 1996, ISSN: 0958-1669.
@article{214,
title = {Local sequence-structure correlations in proteins},
author = { C Bystroff and K T Simons and K F Han and David Baker},
url = {https://www.bakerlab.org/wp-content/uploads/2016/06/bystroff96A.pdf},
issn = {0958-1669},
year = {1996},
date = {1996-08-01},
journal = {Current opinion in biotechnology},
volume = {7},
pages = {417-21},
abstract = {Considerable progress has been made in understanding the relationship between local amino acid sequence and local protein structure. Recent highlights include numerous studies of the structures adopted by short peptides, new approaches to correlating sequence patterns with structure patterns, and folding simulations using simple potentials.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Considerable progress has been made in understanding the relationship between local amino acid sequence and local protein structure. Recent highlights include numerous studies of the structures adopted by short peptides, new approaches to correlating sequence patterns with structure patterns, and folding simulations using simple potentials.
1993
D Baker, A K Shiau, D A Agard
The role of pro regions in protein folding. Journal Article
In: Current Opinion in Cell Biology, vol. 5, pp. 966-70, 1993, ISSN: 0955-0674.
@article{570,
title = {The role of pro regions in protein folding.},
author = { D Baker and A K Shiau and D A Agard},
url = {https://www.bakerlab.org/wp-content/uploads/2016/06/theroleofproregions_Baker1993.pdf},
issn = {0955-0674},
year = {1993},
date = {1993-12-01},
journal = {Current Opinion in Cell Biology},
volume = {5},
pages = {966-70},
abstract = {In vivo, many proteases are synthesized as larger precursors. During the maturation process, the catalytically active protease domain is released from the larger polypeptide or pro-enzyme by one or more proteolytic processing steps. In several well studied cases, amino-terminal pro regions have been shown to play a fundamental role in the folding of the associated protease domains. The mechanism by which pro regions facilitate folding appears to be significantly different from that used by the molecular chaperones. Recent results suggest that the pro region assisted folding mechanism may be used by a wide variety of proteases, and perhaps even by non-proteases.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In vivo, many proteases are synthesized as larger precursors. During the maturation process, the catalytically active protease domain is released from the larger polypeptide or pro-enzyme by one or more proteolytic processing steps. In several well studied cases, amino-terminal pro regions have been shown to play a fundamental role in the folding of the associated protease domains. The mechanism by which pro regions facilitate folding appears to be significantly different from that used by the molecular chaperones. Recent results suggest that the pro region assisted folding mechanism may be used by a wide variety of proteases, and perhaps even by non-proteases.
1992
D Baker, J L Sohl, D A Agard
A protein-folding reaction under kinetic control Journal Article
In: Nature, vol. 356, pp. 263-5, 1992, ISSN: 0028-0836.
@article{329,
title = {A protein-folding reaction under kinetic control},
author = { D Baker and J L Sohl and D A Agard},
issn = {0028-0836},
year = {1992},
date = {1992-03-01},
journal = {Nature},
volume = {356},
pages = {263-5},
abstract = {Synthesis of alpha-lytic protease is as a precursor containing a 166 amino-acid pro region transiently required for the correct folding of the protease domain. By omitting the pro region in an in vitro refolding reaction we trapped an inactive, but folding competent state (I) having an expanded radius yet native-like secondary structure. The I state is stable for weeks at physiological pH in the absence of denaturant, but rapidly folds to the active, native state on addition of the pro region as a separate polypeptide chain. The mechanism of action of the pro region is distinct from that of the chaperonins: rather than reducing the rate of off-pathway reactions, the pro region accelerates the rate-limiting step on the folding pathway by more than 10(7). Because both the I and native states are stable under identical conditions with no detectable interconversion, the folding of alpha-lytic protease must be under kinetic and not thermodynamic control.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Synthesis of alpha-lytic protease is as a precursor containing a 166 amino-acid pro region transiently required for the correct folding of the protease domain. By omitting the pro region in an in vitro refolding reaction we trapped an inactive, but folding competent state (I) having an expanded radius yet native-like secondary structure. The I state is stable for weeks at physiological pH in the absence of denaturant, but rapidly folds to the active, native state on addition of the pro region as a separate polypeptide chain. The mechanism of action of the pro region is distinct from that of the chaperonins: rather than reducing the rate of off-pathway reactions, the pro region accelerates the rate-limiting step on the folding pathway by more than 10(7). Because both the I and native states are stable under identical conditions with no detectable interconversion, the folding of alpha-lytic protease must be under kinetic and not thermodynamic control.