@article{Huang2024b,

title = {Designed endocytosis-inducing proteins degrade targets and amplify signals},

author = {Buwei Huang and Mohamad Abedi and Green Ahn and Brian Coventry and Isaac Sappington and Cong Tang and Rong Wang and Thomas Schlichthaerle and Jason Z. Zhang and Yujia Wang and Inna Goreshnik and Ching Wen Chiu and Adam Chazin-Gray and Sidney Chan and Stacey Gerben and Analisa Murray and Shunzhi Wang and Jason O’Neill and Li Yi and Ronald Yeh and Ayesha Misquith and Anitra Wolf and Luke M. Tomasovic and Dan I. Piraner and Maria J. Duran Gonzalez and Nathaniel R. Bennett and Preetham Venkatesh and Maggie Ahlrichs and Craig Dobbins and Wei Yang and Xinru Wang and Danny D. Sahtoe and Dionne Vafeados and Rubul Mout and Shirin Shivaei and Longxing Cao and Lauren Carter and Lance Stewart and Jamie B. Spangler and Kole T. Roybal and Per Jr Greisen and Xiaochun Li and Gonçalo J. L. Bernardes and Carolyn R. Bertozzi and David Baker},

url = {https://www.nature.com/articles/s41586-024-07948-2, Nature [Open Access] 

https://www.bakerlab.org/wp-content/uploads/2025/03/Huang-et-al-EndoTags-Nature-25Sep2024.pdf, PDF},

doi = {10.1038/s41586-024-07948-2},

year  = {2024},

date = {2024-09-25},

urldate = {2024-09-25},

journal = {Nature},

publisher = {Springer Science and Business Media LLC},

abstract = {Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by endogenous ligands. Therapeutic approaches such as lysosome-targeting chimaeras1,2 (LYTACs) and cytokine receptor-targeting chimeras3 (KineTACs) have used this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. Although powerful, these approaches can be limited by competition with native ligands and requirements for chemical modification that limit genetic encodability and can complicate manufacturing, and, more generally, there may be no native ligands that stimulate endocytosis through a given receptor. Here we describe computational design approaches for endocytosis-triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for insulin-like growth factor 2 receptor (IGF2R) and asialoglycoprotein receptor (ASGPR), sortilin and transferrin receptors, and show that fusing these tags to soluble or transmembrane target protein binders leads to lysosomal trafficking and target degradation. As these receptors have different tissue distributions, the different EndoTags could enable targeting of degradation to different tissues. EndoTag fusion to a PD-L1 antibody considerably increases efficacy in a mouse tumour model compared to antibody alone. The modularity and genetic encodability of EndoTags enables AND gate control for higher-specificity targeted degradation, and the localized secretion of degraders from engineered cells. By promoting endocytosis, EndoTag fusion increases signalling through an engineered ligand–receptor system by nearly 100-fold. EndoTags have considerable therapeutic potential as targeted degradation inducers, signalling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody–drug and antibody–RNA conjugates.



},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Berhanu2024,

title = {Sculpting conducting nanopore size and shape through de novo protein design},

author = {Samuel Berhanu and Sagardip Majumder and Thomas Müntener and James Whitehouse and Carolin Berner and Asim K. Bera and Alex Kang and Binyong Liang and Nasir Khan and Banumathi Sankaran and Lukas K. Tamm and David J. Brockwell and Sebastian Hiller and Sheena E. Radford and David Baker and Anastassia A. Vorobieva},

url = {https://www.science.org/doi/10.1126/science.adn3796, Science},

doi = {10.1126/science.adn3796},

year  = {2024},

date = {2024-07-19},

urldate = {2024-07-19},

journal = {Science},

publisher = {American Association for the Advancement of Science (AAAS)},

abstract = {Transmembrane β-barrels have considerable potential for a broad range of sensing applications. Current engineering approaches for nanopore sensors are limited to naturally occurring channels, which provide suboptimal starting points. By contrast, de novo protein design can in principle create an unlimited number of new nanopores with any desired properties. Here we describe a general approach to designing transmembrane β-barrel pores with different diameters and pore geometries. Nuclear magnetic resonance and crystallographic characterization show that the designs are stably folded with structures resembling those of the design models. The designs have distinct conductances that correlate with their pore diameter, ranging from 110 picosiemens (~0.5 nanometer pore diameter) to 430 picosiemens (~1.1 nanometer pore diameter). Our approach opens the door to the custom design of transmembrane nanopores for sensing and sequencing applications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Peruzzi2024b,

title = {Enhancing extracellular vesicle cargo loading and functional delivery by engineering protein-lipid interactions},

author = {Justin A. Peruzzi and Taylor F. Gunnels and Hailey I. Edelstein and Peilong Lu and David Baker and Joshua N. Leonard and Neha P. Kamat},

url = {https://www.nature.com/articles/s41467-024-49678-z [Nature Communications, Open Access]},

doi = {10.1038/s41467-024-49678-z},

year  = {2024},

date = {2024-07-04},

urldate = {2024-12-00},

journal = {Nature Communications},

publisher = {Springer Science and Business Media LLC},

abstract = {Naturally generated lipid nanoparticles termed extracellular vesicles (EVs) hold significant promise as engineerable therapeutic delivery vehicles. However, active loading of protein cargo into EVs in a manner that is useful for delivery remains a challenge. Here, we demonstrate that by rationally designing proteins to traffic to the plasma membrane and associate with lipid rafts, we can enhance loading of protein cargo into EVs for a set of structurally diverse transmembrane and peripheral membrane proteins. We then demonstrate the capacity of select lipid tags to mediate increased EV loading and functional delivery of an engineered transcription factor to modulate gene expression in target cells. We envision that this technology could be leveraged to develop new EV-based therapeutics that deliver a wide array of macromolecular cargo.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{An2023,

title = {Hallucination of closed repeat proteins containing central pockets},

author = {An L, Hicks DR, Zorine D, Dauparas J, Wicky BIM, Milles LF, Courbet A, Bera AK, Nguyen H, Kang A, Carter L, Baker D},

url = {https://www.nature.com/articles/s41594-023-01112-6, Nature Structural & Molecular Biology [Open Access] },

doi = {10.1038/s41594-023-01112-6},

year  = {2023},

date = {2023-09-28},

urldate = {2023-09-28},

journal = {Nature Structural & Molecular Biology},

abstract = {In pseudocyclic proteins, such as TIM barrels, β barrels, and some helical transmembrane channels, a single subunit is repeated in a cyclic pattern, giving rise to a central cavity that can serve as a pocket for ligand binding or enzymatic activity. Inspired by these proteins, we devised a deep-learning-based approach to broadly exploring the space of closed repeat proteins starting from only a specification of the repeat number and length. Biophysical data for 38 structurally diverse pseudocyclic designs produced in Escherichia coli are consistent with the design models, and the three crystal structures we were able to obtain are very close to the designed structures. Docking studies suggest the diversity of folds and central pockets provide effective starting points for designing small-molecule binders and enzymes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Wang2023,

title = {Improving the secretion of designed protein assemblies through negative design of cryptic transmembrane domains},

author = {Wang, Jing Yang (John)

and Khmelinskaia, Alena

and Sheffler, William

and Miranda, Marcos C.

and Antanasijevic, Aleksandar

and Borst, Andrew J.

and Torres, Susana V.

and Shu, Chelsea

and Hsia, Yang

and Nattermann, Una

and Ellis, Daniel

and Walkey, Carl

and Ahlrichs, Maggie

and Chan, Sidney

and Kang, Alex

and Nguyen, Hannah

and Sydeman, Claire

and Sankaran, Banumathi

and Wu, Mengyu

and Bera, Asim K.

and Carter, Lauren

and Fiala, Brooke

and Murphy, Michael

and Baker, David

and Ward, Andrew B.

and King, Neil P.},

url = {https://www.pnas.org/doi/10.1073/pnas.2214556120, PNAS (Open Access)},

doi = {10.1073/pnas.2214556120},

year  = {2023},

date = {2023-03-08},

urldate = {2023-03-08},

journal = {Proceedings of the National Academy of Sciences},

abstract = {Computationally designed protein nanoparticles have recently emerged as a promising platform for the development of new vaccines and biologics. For many applications, secretion of designed nanoparticles from eukaryotic cells would be advantageous, but in practice, they often secrete poorly. Here we show that designed hydrophobic interfaces that drive nanoparticle assembly are often predicted to form cryptic transmembrane domains, suggesting that interaction with the membrane insertion machinery could limit efficient secretion. We develop a general computational protocol, the Degreaser, to design away cryptic transmembrane domains without sacrificing protein stability. The retroactive application of the Degreaser to previously designed nanoparticle components and nanoparticles considerably improves secretion, and modular integration of the Degreaser into design pipelines results in new nanoparticles that secrete as robustly as naturally occurring protein assemblies. Both the Degreaser protocol and the nanoparticles we describe may be broadly useful in biotechnological applications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Vorobieva2021,

title = {De novo design of transmembrane beta barrels},

author = {Vorobieva, Anastassia A. and White, Paul and Liang, Binyong and Horne, Jim E. and Bera, Asim K. and Chow, Cameron M. and Gerben, Stacey and Marx, Sinduja and Kang, Alex and Stiving, Alyssa Q. and Harvey, Sophie R. and Marx, Dagan C. and Khan, G. Nasir and Fleming, Karen G. and Wysocki, Vicki H. and Brockwell, David J. and Tamm, Lukas K. and Radford, Sheena E. and Baker, David},

url = {https://science.sciencemag.org/content/371/6531/eabc8182, Science

https://www.bakerlab.org/wp-content/uploads/2021/02/Vorobieva_etal_Science2021_De_Novo_Transmembrane_beta_barrels.pdf, Download PDF},

doi = {10.1126/science.abc8182},

year  = {2021},

date = {2021-02-19},

urldate = {2021-02-19},

journal = {Science},

volume = {371},

number = {6531},

abstract = {Computational design offers the possibility of making proteins with customized structures and functions. The range of accessible protein scaffolds has expanded with the design of increasingly complex cytoplasmic proteins and, recently, helical membrane proteins. Vorobieva et al. describe the successful computational design of eight-stranded transmembrane β-barrel proteins (TMBs). Using an iterative approach, they show the importance of negative design to prevent off-target structures and gain insight into the sequence determinants of TMB folding. Twenty-three designs satisfied biochemical screens for a TMB structure, and two structures were experimentally validated by nuclear magnetic resonance spectroscopy or x-ray crystallography. This is a step toward the custom design of pores for applications such as single-molecule sequencing.Science, this issue p. eabc8182INTRODUCTIONDespite their key biological roles, only a few proteins that fold into lipid membranes have been designed de novo. A class of membrane proteins{textemdash}transmembrane β barrels (TMBs){textemdash}forms a continuous sheet that closes on itself in lipid membranes. In addition to the challenge of designing β-sheet proteins, which are prone to misfolding and aggregation if folding is not properly controlled, the computational design of TMBs is complicated by limited understanding of TMB folding. As a result, no TMB has been designed de novo to date.Although the folding of TMBs in vivo is catalyzed by the β-barrel assembly machinery (BAM), many TMBs can also fold spontaneously in synthetic membranes to form stable pores, making them attractive for biotechnology and single-molecule analytical applications. Hence, de novo design of TMBs has potential both for understanding the determinants of TMB folding and membrane insertion and for the custom engineering of TMB nanopores.RATIONALEWe used de novo protein design to distill key principles of TMB folding through several design-build-test cycles. We iterated between hypothesis formulation, its implementation into computational design methods, and experimental characterization of the resulting proteins. To focus on the fundamental principles of TMB folding in the absence of complications due to interactions with chaperones and BAM in vivo, we focused on the challenge of de novo design of eight-stranded TMBs, which can fold and assemble into synthetic lipid membranes.RESULTSWe used a combination of purely geometric models and explicit Rosetta protein structure simulations to determine the constraints that β-strand connectivity and membrane embedding place on the TMB architecture. Through a series of design-build-test cycles, we found that, unlike almost all other classes of proteins, locally destabilizing sequences are critical for expression and folding of TMBs, and that the β-turns that translocate through the bilayer during folding have to be destabilized to enable correct assembly in the membrane. Our results suggest that premature formation of β hairpins may result in off-target β-sheet structures that compete with proper membrane insertion and folding, and hence the β hairpins of TMBs must be designed such that they are only transiently formed prior to membrane insertion, when the protein is in an aqueous environment. In the hydrophobic environment of the lipid bilayer, the full TMB can assemble because the membrane-facing nonpolar residues, which would tend to cluster nonspecifically in an aqueous environment, instead make favorable interactions with the lipids. As the TMB assembles, the β hairpins are stabilized by interactions with the neighboring β strands.Using computational methods that incorporate the above insights, we designed TMB sequences that successfully fold and assemble into both detergent micelles and lipid bilayers. Two of the designs were highly stable and could fold into liposomes more rapidly and reversibly than the transmembrane domain of the model outer membrane protein A (tOmpA) of Escherichia coli. A nuclear magnetic resonance solution structure and a high-resolution crystal structure for two different designs closely match the design models, showing that the TMB design method developed here can generate new structures with atomic-level accuracy.CONCLUSIONThis study elucidates key principles for de novo design of transmembrane β barrels, ranging from constraints on β-barrel architecture and β-hairpin design, as well as local and global sequence features. Our designs provide starting points for the bottom-up elucidation of the molecular mechanisms underlying TMB folding and interactions with the cellular outer membrane folding and insertion machinery. More generally, our work demonstrates that TMBs can be designed with atomic-level accuracy and opens the door to custom design of nanopores tailored for applications such as single-molecule sensing and sequencing.De novo{textendash}designed eight-stranded transmembrane β barrels fold spontaneously and reversibly into synthetic lipid membranes.The illustration shows the crystal structure of the protein TMB2.17 designed in this study, which adopts a structure identical to the design model.Credit: Ian Haydon.Transmembrane β-barrel proteins (TMBs) are of great interest for single-molecule analytical technologies because they can spontaneously fold and insert into membranes and form stable pores, but the range of pore properties that can be achieved by repurposing natural TMBs is limited. We leverage the power of de novo computational design coupled with a {textquotedblleft}hypothesis, design, and test{textquotedblright} approach to determine TMB design principles, notably, the importance of negative design to slow β-sheet assembly. We design new eight-stranded TMBs, with no homology to known TMBs, that insert and fold reversibly into synthetic lipid membranes and have nuclear magnetic resonance and x-ray crystal structures very similar to the computational models. These advances should enable the custom design of pores for a wide range of applications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@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}

}

@article{Lu1042,

title = {Accurate computational design of multipass transmembrane proteins},

author = {Lu, Peilong and Min, Duyoung and DiMaio, Frank and Wei, Kathy Y. and Vahey, Michael D. and Boyken, Scott E. and Chen, Zibo and Fallas, Jorge A. and Ueda, George and Sheffler, William and Mulligan, Vikram Khipple and Xu, Wenqing and Bowie, James U. and Baker, David},

url = {http://science.sciencemag.org/content/359/6379/1042

https://www.bakerlab.org/wp-content/uploads/2018/03/Lu_Science_2018.pdf},

doi = {10.1126/science.aaq1739},

issn = {0036-8075},

year  = {2018},

date = {2018-03-02},

journal = {Science},

volume = {359},

number = {6379},

pages = {1042--1046},

abstract = {In recent years, soluble protein design has achieved successes such as artificial enzymes and large protein cages. Membrane proteins present a considerable design challenge, but here too there have been advances, including the design of a zinc-transporting tetramer. Lu et al. report the design of stable transmembrane monomers, homodimers, trimers, and tetramers with up to eight membrane-spanning regions in an oligomer. The designed proteins adopted the target oligomerization state and localized to the predicted cellular membranes, and crystal structures of the designed dimer and tetramer reflected the design models.Science, this issue p. 1042The computational design of transmembrane proteins with more than one membrane-spanning region remains a major challenge. We report the design of transmembrane monomers, homodimers, trimers, and tetramers with 76 to 215 residue subunits containing two to four membrane-spanning regions and up to 860 total residues that adopt the target oligomerization state in detergent solution. The designed proteins localize to the plasma membrane in bacteria and in mammalian cells, and magnetic tweezer unfolding experiments in the membrane indicate that they are very stable. Crystal structures of the designed dimer and tetramer{textemdash}a rocket-shaped structure with a wide cytoplasmic base that funnels into eight transmembrane helices{textemdash}are very close to the design models. Our results pave the way for the design of multispan membrane proteins with new functions.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{608,

title = {The modular structure of the inner-membrane ring component PrgK facilitates assembly of the type III secretion system basal body},

author = { Julien R C Bergeron and Liam J Worrall and Soumya De and Nikolaos G Sgourakis and Adrienne H Cheung and Emilie Lameignere and Mark Okon and Gregory A Wasney and David Baker and Lawrence P McIntosh and Natalie C J Strynadka},

url = {http://www.bakerlab.org/wp-content/uploads/2015/12/themodularstructure_Baker2015.pdf},

doi = {10.1016/j.str.2014.10.021},

issn = {1878-4186},

year  = {2015},

date = {2015-01-01},

journal = {Structure (London, England : 1993)},

volume = {23},

pages = {161-72},

abstract = {The type III secretion system (T3SS) is a large macromolecular assembly found at the surface of many pathogenic Gram-negative bacteria. Its role is to inject toxic "effector" proteins into the cells of infected organisms. The molecular details of the assembly of this large, multimembrane-spanning complex remain poorly understood. Here, we report structural, biochemical, and functional analyses of PrgK, an inner-membrane component of the prototypical Salmonella typhimurium T3SS. We have obtained the atomic structures of the two ring building globular domains and show that the C-terminal transmembrane helix is not essential for assembly and secretion. We also demonstrate that structural rearrangement of the two PrgK globular domains, driven by an interconnecting linker region, may promote oligomerization into ring structures. Finally, we used electron microscopy-guided symmetry modeling to propose a structural model for the intimately associated PrgH-PrgK ring interaction within the assembled basal body.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{622,

title = {High-resolution modeling of transmembrane helical protein structures from distant homologues.},

author = { Kuang-Yui M Chen and Jiaming Sun and Jason S Salvo and David Baker and Patrick Barth},

url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Chen_PLOS_2014.pdf},

doi = {10.1371/journal.pcbi.1003636},

issn = {1553-7358},

year  = {2014},

date = {2014-05-01},

journal = {PLoS computational biology},

volume = {10},

pages = {e1003636},

abstract = {Eukaryotic transmembrane helical (TMH) proteins perform a wide diversity of critical cellular functions, but remain structurally largely uncharacterized and their high-resolution structure prediction is currently hindered by the lack of close structural homologues. To address this problem, we present a novel and generic method for accurately modeling large TMH protein structures from distant homologues exhibiting distinct loop and TMH conformations. Models of the adenosine A2AR and chemokine CXCR4 receptors were first ranked in GPCR-DOCK blind prediction contests in the receptor structure accuracy category. In a benchmark of 50 TMH protein homolog pairs of diverse topology (from 5 to 12 TMHs), size (from 183 to 420 residues) and sequence identity (from 15% to 70%), the method improves most starting templates, and achieves near-atomic accuracy prediction of membrane-embedded regions. Unlike starting templates, the models are of suitable quality for computer-based protein engineering: redesigned models and redesigned X-ray structures exhibit very similar native interactions. The method should prove useful for the atom-level modeling and design of a large fraction of structurally uncharacterized TMH proteins from a wide range of structural homologues.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{433,

title = {Structural basis for gating charge movement in the voltage sensor of a sodium channel},

author = { Vladimir Yarov-Yarovoy and Paul G DeCaen and Ruth E Westenbroek and Chien-Yuan Pan and Todd Scheuer and David Baker and William A Catterall},

url = {https://www.bakerlab.org/wp-content/uploads/2018/06/1.full_.pdf

http://www.pnas.org/content/109/2/E93/1},

doi = {10.1073/pnas.1118434109},

issn = {1091-6490},

year  = {2012},

date = {2012-01-01},

journal = {Proceedings of the National Academy of Sciences of the United States of America},

volume = {109},

pages = {E93-102},

abstract = {Voltage-dependent gating of ion channels is essential for electrical signaling in excitable cells, but the structural basis for voltage sensor function is unknown. We constructed high-resolution structural models of resting, intermediate, and activated states of the voltage-sensing domain of the bacterial sodium channel NaChBac using the Rosetta modeling method, crystal structures of related channels, and experimental data showing state-dependent interactions between the gating charge-carrying arginines in the S4 segment and negatively charged residues in neighboring transmembrane segments. The resulting structural models illustrate a network of ionic and hydrogen-bonding interactions that are made sequentially by the gating charges as they move out under the influence of the electric field. The S4 segment slides 6-8 r A outward through a narrow groove formed by the S1, S2, and S3 segments, rotates ~30textdegree, and tilts sideways at a pivot point formed by a highly conserved hydrophobic region near the middle of the voltage sensor. The S4 segment has a 3(10)-helical conformation in the narrow inner gating pore, which allows linear movement of the gating charges across the inner one-half of the membrane. Conformational changes of the intracellular one-half of S4 during activation are rigidly coupled to lateral movement of the S4-S5 linker, which could induce movement of the S5 and S6 segments and open the intracellular gate of the pore. We confirmed the validity of these structural models by comparing with a high-resolution structure of a NaChBac homolog and showing predicted molecular interactions of hydrophobic residues in the S4 segment in disulfide-locking studies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{594,

title = {Design and characterization of stabilized derivatives of human CD4D12 and CD4D1},

author = { Piyali Saha and Bipasha Barua and Sanchari Bhattacharyya and M M Balamurali and William R Schief and David Baker and Raghavan Varadarajan},

url = {https://www.bakerlab.org/wp-content/uploads/2018/06/bi200870r.pdf

https://pubs.acs.org/doi/abs/10.1021/bi200870r},

doi = {10.1021/bi200870r},

issn = {1520-4995},

year  = {2011},

date = {2011-09-01},

journal = {Biochemistry},

volume = {50},

pages = {7891-900},

abstract = {CD4 is present on the surface of T-lymphocytes and is the primary cellular receptor for HIV-1. CD4 consists of a cytoplasmic tail, one transmembrane region, and four extracellular domains, D1-D4. A construct consisting of the first two domains of CD4 (CD4D12) is folded and binds gp120 with similar affinity as soluble 4-domain CD4 (sCD4). However, the first domain alone (CD4D1) was previously shown to be largely unfolded and had 3-fold weaker affinity for gp120 when compared to sCD4 [Sharma, D.; et al. (2005) Biochemistry 44, 16192-16202]. We now report the design and characterization of three single-site mutants of CD4D12 (G6A, L51I, and V86L) and one multisite mutant of CD4D1 (G6A/L51I/L5K/F98T). G6A, L51I, and V86L are cavity-filling mutations while L5K and F98T are surface mutations which were introduced to minimize the aggregation of CD4D1 upon removal of the second domain. Two mutations, G6A and V86L in CD4D12 increased the stability and yield of the protein relative to the wild-type protein. The mutant CD4D1 (CD4D1a) with the 4 mutations was folded and more stable compared to the original CD4D1, but both bound gp120 with comparable affinity. In in vitro neutralization assays, both CD4D1a and G6A-CD4D12 were able to neutralize diverse HIV-1 viruses with similar IC(50)s as 4-domain CD4. These stabilized derivatives of human CD4 can be useful starting points for the design of other more complex viral entry inhibitors.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{261,

title = {Interactions of the Transmembrane Polymeric Rings of the Salmonella enterica Serovar Typhimurium Type III Secretion System},

author = { Sarah Sanowar and Pragya Singh and Richard A Pfuetzner and Ingemar Andr'e and Hongjin Zheng and Thomas Spreter and Natalie C J Strynadka and Tamir Gonen and David Baker and David R Goodlett and Samuel I Miller},

issn = {2150-7511},

year  = {2010},

date = {2010-00-01},

journal = {mBio},

volume = {1},

abstract = {The type III secretion system (T3SS) is an interspecies protein transport machine that plays a major role in interactions of Gram-negative bacteria with animals and plants by delivering bacterial effector proteins into host cells. T3SSs span both membranes of Gram-negative bacteria by forming a structure of connected oligomeric rings termed the needle complex (NC). Here, the localization of subunits in the Salmonella enterica serovar Typhimurium T3SS NC were probed via mass spectrometry-assisted identification of chemical cross-links in intact NC preparations. Cross-links between amino acids near the amino terminus of the outer membrane ring component InvG and the carboxyl terminus of the inner membrane ring component PrgH and between the two inner membrane components PrgH and PrgK allowed for spatial localization of the three ring components within the electron density map structures of NCs. Mutational and biochemical analysis demonstrated that the amino terminus of InvG and the carboxyl terminus of PrgH play a critical role in the assembly and function of the T3SS apparatus. Analysis of an InvG mutant indicates that the structure of the InvG oligomer can affect the switching of the T3SS substrate to translocon and effector components. This study provides insights into how structural organization of needle complex base components promotes T3SS assembly and function.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{139,

title = {The structure of a receptor with two associating transmembrane domains on the cell surface: integrin alphaIIbbeta3},

author = { Jieqing Zhu and Bing-Hao Luo and Patrick Barth and Jack Schonbrun and David Baker and Timothy A Springer},

issn = {1097-4164},

year  = {2009},

date = {2009-04-01},

journal = {Molecular cell},

volume = {34},

pages = {234-49},

abstract = {Structures of intact receptors with single-pass transmembrane domains are essential to understand how extracellular and cytoplasmic domains regulate association and signaling through transmembrane domains. A chemical and computational method to determine structures of the membrane regions of such receptors on the cell surface is developed here and validated with glycophorin A. An integrin heterodimer structure reveals association over most of the lengths of the alpha and beta transmembrane domains and shows that the principles governing association of hetero and homo transmembrane dimers differ. A turn at the Gly of the juxtamembrane GFFKR motif caps the alpha TM helix and brings the two Phe of GFFKR into the alpha/beta interface. A juxtamembrane Lys residue in beta also has an important role in the interface. The structure shows how transmembrane association/dissociation regulates integrin signaling. A joint ectodomain and membrane structure shows that substantial flexibility between the extracellular and TM domains is compatible with TM signaling.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{132,

title = {Rationally designed integrin beta3 mutants stabilized in the high affinity conformation},

author = { Bing-Hao Luo and John Karanicolas and Laura D Harmacek and David Baker and Timothy A Springer},

issn = {0021-9258},

year  = {2009},

date = {2009-02-01},

journal = {The Journal of biological chemistry},

volume = {284},

pages = {3917-24},

abstract = {Integrins are important cell surface receptors that transmit bidirectional signals across the membrane. It has been shown that a conformational change of the integrin beta-subunit headpiece (i.e. the beta I domain and the hybrid domain) plays a critical role in regulating integrin ligand binding affinity and function. Previous studies have used coarse methods (a glycan wedge, mutations in transmembrane contacts) to force the beta-subunit into either the open or closed conformation. Here, we demonstrate a detailed understanding of this conformational change by applying computational design techniques to select five amino acid side chains that play an important role in the energetic balance between the open and closed conformations of alphaIIbbeta3. Eight single-point mutants were designed at these sites, of which five bound ligands much better than wild type. Further, these mutants were found to be in a more extended conformation than wild type, suggesting that the conformational change at the ligand binding headpiece was propagated to the legs of the integrin. This detailed understanding of the conformational change will assist in the development of allosteric drugs that either stabilize or destabilize specific integrin conformations without occluding the ligand-binding site.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{120,

title = {Toward high-resolution prediction and design of transmembrane helical protein structures},

author = { P Barth and J Schonbrun and David Baker},

url = {https://www.bakerlab.org/wp-content/uploads/2016/08/barth07A.pdf},

issn = {0027-8424},

year  = {2007},

date = {2007-10-01},

journal = {Proceedings of the National Academy of Sciences of the United States of America},

volume = {104},

pages = {15682-7},

abstract = {The prediction and design at the atomic level of membrane protein structures and interactions is a critical but unsolved challenge. To address this problem, we have developed an all-atom physical model that describes intraprotein and protein-solvent interactions in the membrane environment. We evaluated the ability of the model to recapitulate the energetics and structural specificities of polytopic membrane proteins by using a battery of in silico prediction and design tests. First, in side-chain packing and design tests, the model successfully predicts the side-chain conformations at 73% of nonexposed positions and the native amino acid identities at 34% of positions in naturally occurring membrane proteins. Second, the model predicts significant energy gaps between native and nonnative structures of transmembrane helical interfaces and polytopic membrane proteins. Third, distortions in transmembrane helices are successfully recapitulated in docking experiments by using fragments of ideal helices judiciously defined around helical kinks. Finally, de novo structure prediction reaches near-atomic accuracy (<2.5 A) for several small membrane protein domains (<150 residues). The success of the model highlights the critical role of van der Waals and hydrogen-bonding interactions in the stability and structural specificity of membrane protein structures and sets the stage for the high-resolution prediction and design of complex membrane protein architectures.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{165,

title = {Multipass membrane protein structure prediction using Rosetta},

author = { Vladimir Yarov-Yarovoy and Jack Schonbrun and David Baker},

url = {https://www.bakerlab.org/wp-content/uploads/2016/08/yarov-yarovoy06B.pdf},

issn = {1097-0134},

year  = {2006},

date = {2006-03-01},

journal = {Proteins},

volume = {62},

pages = {1010-25},

abstract = {We describe the adaptation of the Rosetta de novo structure prediction method for prediction of helical transmembrane protein structures. The membrane environment is modeled by embedding the protein chain into a model membrane represented by parallel planes defining hydrophobic, interface, and polar membrane layers for each energy evaluation. The optimal embedding is determined by maximizing the exposure of surface hydrophobic residues within the membrane and minimizing hydrophobic exposure outside of the membrane. Protein conformations are built up using the Rosetta fragment assembly method and evaluated using a new membrane-specific version of the Rosetta low-resolution energy function in which residue-residue and residue-environment interactions are functions of the membrane layer in addition to amino acid identity, distance, and density. We find that lower energy and more native-like structures are achieved by sequential addition of helices to a growing chain, which may mimic some aspects of helical protein biogenesis after translocation, rather than folding the whole chain simultaneously as in the Rosetta soluble protein prediction method. In tests on 12 membrane proteins for which the structure is known, between 51 and 145 residues were predicted with root-mean-square deviation <4 A from the native structure.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}