Preprints are available on bioRxiv.
1.
Sahtoe, Danny D.; Coscia, Adrian; Mustafaoglu, Nur; Miller, Lauren M.; Olal, Daniel; Vulovic, Ivan; Yu, Ta-Yi; Goreshnik, Inna; Lin, Yu-Ru; Clark, Lars; Busch, Florian; Stewart, Lance; Wysocki, Vicki H.; Ingber, Donald E.; Abraham, Jonathan; Baker, David
Transferrin receptor targeting by de novo sheet extension Journal Article
In: Proceedings of the National Academy of Sciences, 2021.
@article{Sahtoe2021,
title = {Transferrin receptor targeting by de novo sheet extension},
author = {Sahtoe, Danny D. and Coscia, Adrian and Mustafaoglu, Nur and Miller, Lauren M. and Olal, Daniel and Vulovic, Ivan and Yu, Ta-Yi and Goreshnik, Inna and Lin, Yu-Ru and Clark, Lars and Busch, Florian and Stewart, Lance and Wysocki, Vicki H. and Ingber, Donald E. and Abraham, Jonathan and Baker, David},
url = {https://www.pnas.org/content/118/17/e2021569118, PNAS
},
doi = {10.1073/pnas.2021569118},
year = {2021},
date = {2021-04-27},
urldate = {2021-04-27},
journal = {Proceedings of the National Academy of Sciences},
abstract = {The de novo design of proteins that bind natural target proteins is useful for a variety of biomedical and biotechnological applications. We describe a design strategy to target proteins containing an exposed beta edge strand. We use the approach to design binders to the human transferrin receptor which shuttles back and forth across the blood{textendash}brain barrier. Such binders could be useful for the delivery of therapeutics into the brain.The de novo design of polar protein{textendash}protein interactions is challenging because of the thermodynamic cost of stripping water away from the polar groups. Here, we describe a general approach for designing proteins which complement exposed polar backbone groups at the edge of beta sheets with geometrically matched beta strands. We used this approach to computationally design small proteins that bind to an exposed beta sheet on the human transferrin receptor (hTfR), which shuttles interacting proteins across the blood{textendash}brain barrier (BBB), opening up avenues for drug delivery into the brain. We describe a design which binds hTfR with a 20 nM Kd, is hyperstable, and crosses an in vitro microfluidic organ-on-a-chip model of the human BBB. Our design approach provides a general strategy for creating binders to protein targets with exposed surface beta edge strands.Crystal structures have been deposited in the RCSB PDB with the accession nos. 6WRX, 6WRW, and 6WRV. Additional supporting data has been deposited in the online Zenodo repository (https://zenodo.org/record/4594115) (47). All other study data are included in the article and/or supporting information.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The de novo design of proteins that bind natural target proteins is useful for a variety of biomedical and biotechnological applications. We describe a design strategy to target proteins containing an exposed beta edge strand. We use the approach to design binders to the human transferrin receptor which shuttles back and forth across the blood{textendash}brain barrier. Such binders could be useful for the delivery of therapeutics into the brain.The de novo design of polar protein{textendash}protein interactions is challenging because of the thermodynamic cost of stripping water away from the polar groups. Here, we describe a general approach for designing proteins which complement exposed polar backbone groups at the edge of beta sheets with geometrically matched beta strands. We used this approach to computationally design small proteins that bind to an exposed beta sheet on the human transferrin receptor (hTfR), which shuttles interacting proteins across the blood{textendash}brain barrier (BBB), opening up avenues for drug delivery into the brain. We describe a design which binds hTfR with a 20 nM Kd, is hyperstable, and crosses an in vitro microfluidic organ-on-a-chip model of the human BBB. Our design approach provides a general strategy for creating binders to protein targets with exposed surface beta edge strands.Crystal structures have been deposited in the RCSB PDB with the accession nos. 6WRX, 6WRW, and 6WRV. Additional supporting data has been deposited in the online Zenodo repository (https://zenodo.org/record/4594115) (47). All other study data are included in the article and/or supporting information.
2.
Butterfield, Gabriel L. *; Lajoie, Marc J. *; Gustafson, Heather H.; Sellers, Drew L.; Nattermann, Una; Ellis, Daniel; Bale, Jacob B.; Ke, Sharon; Lenz, Garreck H.; Yehdego, Angelica; Ravichandran, Rashmi; Pun, Suzie H.; King, Neil P.; 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.
3.
Hsia*, Yang; Bale*, Jacob B.; Gonen, Shane; Shi, Dan; Sheffler, William; Fong, Kimberly K.; Nattermann,; Xu, Chunfu; Huang, Po-Ssu; Ravichandran, Rashmi; Yi, Sue; Davis, Trisha N.; Gonen, Tamir; King, Neil P.; Baker, David
Design of a hyperstable 60-subunit protein icosahedron Journal Article
In: Nature, 2016.
@article{Hsia2016,
title = {Design of a hyperstable 60-subunit protein icosahedron},
author = { Yang Hsia* and Jacob B. Bale* and Shane Gonen and Dan Shi and William Sheffler and Kimberly K. Fong and Nattermann and Chunfu Xu and Po-Ssu Huang and Rashmi Ravichandran and Sue Yi and Trisha N. Davis and Tamir Gonen and Neil P. King and David Baker},
url = {https://www.bakerlab.org/wp-content/uploads/2016/06/Hsia_Nature_2016.pdf},
doi = {10.1038/nature18010},
year = {2016},
date = {2016-06-15},
journal = {Nature},
abstract = {The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/ exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/ exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.
4.
Procko, Erik; Berguig, Geoffrey Y; Shen, Betty W; Song, Yifan; Frayo, Shani; Convertine, Anthony J; Margineantu, Daciana; Booth, Garrett; Correia, Bruno E; Cheng, Yuanhua; Schief, William R; Hockenbery, David M; Press, Oliver W; Stoddard, Barry L; Stayton, Patrick S; Baker, David
A computationally designed inhibitor of an epstein-barr viral bcl-2 protein induces apoptosis in infected cells. Journal Article
In: Cell, vol. 157, pp. 1644-56, 2014, ISSN: 1097-4172.
@article{538,
title = {A computationally designed inhibitor of an epstein-barr viral bcl-2 protein induces apoptosis in infected cells.},
author = { Erik Procko and Geoffrey Y Berguig and Betty W Shen and Yifan Song and Shani Frayo and Anthony J Convertine and Daciana Margineantu and Garrett Booth and Bruno E Correia and Yuanhua Cheng and William R Schief and David M Hockenbery and Oliver W Press and Barry L Stoddard and Patrick S Stayton and David Baker},
url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Procko2014A.pdf},
doi = {10.1016/j.cell.2014.04.034},
issn = {1097-4172},
year = {2014},
date = {2014-06-01},
journal = {Cell},
volume = {157},
pages = {1644-56},
abstract = {Because apoptosis of infected cells can limit virus production and spread, some viruses have co-opted prosurvival genes from the host. This includes the Epstein-Barr virus (EBV) gene BHRF1, a homolog of human Bcl-2 proteins that block apoptosis and are associated with cancer. Computational design and experimental optimization were used to generate a novel protein called BINDI that binds BHRF1 with picomolar affinity. BINDI recognizes the hydrophobic cleft of BHRF1 in a manner similar to other Bcl-2 protein interactions but makes many additional contacts to achieve exceptional affinity and specificity. BINDI induces apoptosis in EBV-infected cancer lines, and when delivered with an antibody-targeted intracellular delivery carrier, BINDI suppressed tumor growth and extended survival in a xenograft disease model of EBV-positive human lymphoma. High-specificity-designed proteins that selectively kill target cells may provide an advantage over the toxic compounds used in current generation antibody-drug conjugates.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Because apoptosis of infected cells can limit virus production and spread, some viruses have co-opted prosurvival genes from the host. This includes the Epstein-Barr virus (EBV) gene BHRF1, a homolog of human Bcl-2 proteins that block apoptosis and are associated with cancer. Computational design and experimental optimization were used to generate a novel protein called BINDI that binds BHRF1 with picomolar affinity. BINDI recognizes the hydrophobic cleft of BHRF1 in a manner similar to other Bcl-2 protein interactions but makes many additional contacts to achieve exceptional affinity and specificity. BINDI induces apoptosis in EBV-infected cancer lines, and when delivered with an antibody-targeted intracellular delivery carrier, BINDI suppressed tumor growth and extended survival in a xenograft disease model of EBV-positive human lymphoma. High-specificity-designed proteins that selectively kill target cells may provide an advantage over the toxic compounds used in current generation antibody-drug conjugates.
5.
Boissel, Sandrine; Jarjour, Jordan; Astrakhan, Alexander; Adey, Andrew; Gouble, Agn`es; Duchateau, Philippe; Shendure, Jay; Stoddard, Barry L; Certo, Michael T; Baker, David; Scharenberg, Andrew M
megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering. Journal Article
In: Nucleic acids research, 2013, ISSN: 1362-4962.
@article{516,
title = {megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering.},
author = { Sandrine Boissel and Jordan Jarjour and Alexander Astrakhan and Andrew Adey and Agn`es Gouble and Philippe Duchateau and Jay Shendure and Barry L Stoddard and Michael T Certo and David Baker and Andrew M Scharenberg},
url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Boissel_NAR_2013.pdf},
issn = {1362-4962},
year = {2013},
date = {2013-11-01},
journal = {Nucleic acids research},
abstract = {Rare-cleaving endonucleases have emerged as important tools for making targeted genome modifications. While multiple platforms are now available to generate reagents for research applications, each existing platform has significant limitations in one or more of three key properties necessary for therapeutic application: efficiency of cleavage at the desired target site, specificity of cleavage (i.e. rate of cleavage at textquoterightoff-targettextquoteright sites), and efficient/facile means for delivery to desired target cells. Here, we describe the development of a single-chain rare-cleaving nuclease architecture, which we designate textquoterightmegaTALtextquoteright, in which the DNA binding region of a transcription activator-like (TAL) effector is used to textquoterightaddresstextquoteright a site-specific meganuclease adjacent to a single desired genomic target site. This architecture allows the generation of extremely active and hyper-specific compact nucleases that are compatible with all current viral and nonviral cell delivery methods.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Rare-cleaving endonucleases have emerged as important tools for making targeted genome modifications. While multiple platforms are now available to generate reagents for research applications, each existing platform has significant limitations in one or more of three key properties necessary for therapeutic application: efficiency of cleavage at the desired target site, specificity of cleavage (i.e. rate of cleavage at textquoterightoff-targettextquoteright sites), and efficient/facile means for delivery to desired target cells. Here, we describe the development of a single-chain rare-cleaving nuclease architecture, which we designate textquoterightmegaTALtextquoteright, in which the DNA binding region of a transcription activator-like (TAL) effector is used to textquoterightaddresstextquoteright a site-specific meganuclease adjacent to a single desired genomic target site. This architecture allows the generation of extremely active and hyper-specific compact nucleases that are compatible with all current viral and nonviral cell delivery methods.
6.
Bergeron, Julien R C; Worrall, Liam J; Sgourakis, Nikolaos G; DiMaio, Frank; Pfuetzner, Richard A; Felise, Heather B; Vuckovic, Marija; Yu, Angel C; Miller, Samuel I; Baker, David; Strynadka, Natalie C J
A Refined Model of the Prototypical Salmonella SPI-1 T3SS Basal Body Reveals the Molecular Basis for Its Assembly Journal Article
In: PLoS pathogens, vol. 9, pp. e1003307, 2013, ISSN: 1553-7374.
@article{468,
title = {A Refined Model of the Prototypical Salmonella SPI-1 T3SS Basal Body Reveals the Molecular Basis for Its Assembly},
author = { Julien R C Bergeron and Liam J Worrall and Nikolaos G Sgourakis and Frank DiMaio and Richard A Pfuetzner and Heather B Felise and Marija Vuckovic and Angel C Yu and Samuel I Miller and David Baker and Natalie C J Strynadka},
url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Bergeron_ppat1003307_13T.pdf},
doi = {10.1371/journal.ppat.1003307},
issn = {1553-7374},
year = {2013},
date = {2013-04-01},
journal = {PLoS pathogens},
volume = {9},
pages = {e1003307},
abstract = {The T3SS injectisome is a syringe-shaped macromolecular assembly found in pathogenic Gram-negative bacteria that allows for the direct delivery of virulence effectors into host cells. It is composed of a "basal body", a lock-nut structure spanning both bacterial membranes, and a "needle" that protrudes away from the bacterial surface. A hollow channel spans throughout the apparatus, permitting the translocation of effector proteins from the bacterial cytosol to the host plasma membrane. The basal body is composed largely of three membrane-embedded proteins that form oligomerized concentric rings. Here, we report the crystal structures of three domains of the prototypical Salmonella SPI-1 basal body, and use a new approach incorporating symmetric flexible backbone docking and EM data to produce a model for their oligomeric assembly. The obtained models, validated by biochemical and in vivo assays, reveal the molecular details of the interactions driving basal body assembly, and notably demonstrate a conserved oligomerization mechanism.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The T3SS injectisome is a syringe-shaped macromolecular assembly found in pathogenic Gram-negative bacteria that allows for the direct delivery of virulence effectors into host cells. It is composed of a "basal body", a lock-nut structure spanning both bacterial membranes, and a "needle" that protrudes away from the bacterial surface. A hollow channel spans throughout the apparatus, permitting the translocation of effector proteins from the bacterial cytosol to the host plasma membrane. The basal body is composed largely of three membrane-embedded proteins that form oligomerized concentric rings. Here, we report the crystal structures of three domains of the prototypical Salmonella SPI-1 basal body, and use a new approach incorporating symmetric flexible backbone docking and EM data to produce a model for their oligomeric assembly. The obtained models, validated by biochemical and in vivo assays, reveal the molecular details of the interactions driving basal body assembly, and notably demonstrate a conserved oligomerization mechanism.
7.
Payne, G S; Baker, D; van Tuinen, E; Schekman, R
Protein transport to the vacuole and receptor-mediated endocytosis by clathrin heavy chain-deficient yeast Journal Article
In: The Journal of cell biology, vol. 106, pp. 1453-61, 1988, ISSN: 0021-9525.
@article{333,
title = {Protein transport to the vacuole and receptor-mediated endocytosis by clathrin heavy chain-deficient yeast},
author = { G S Payne and D Baker and E van Tuinen and R Schekman},
url = {https://www.bakerlab.org/wp-content/uploads/2016/06/payne88A.pdf},
issn = {0021-9525},
year = {1988},
date = {1988-05-01},
journal = {The Journal of cell biology},
volume = {106},
pages = {1453-61},
abstract = {Clathrin heavy chain-deficient mutants (chcl) of Saccharomyces cerevisiae are viable but exhibit compromised growth rates. To investigate the role of clathrin in intercompartmental protein transport, two pathways have been monitored in chcl cells: transport of newly synthesized vacuolar proteins to the vacuole and receptor-mediated uptake of mating pheromone. Newly synthesized precursors of the vacuolar protease carboxypeptidase Y (CPY) were converted to mature CPY with similar kinetics in mutant and wild-type cells. chcl cells did not aberrantly secrete CPY and immunolocalization techniques revealed most of the CPY in chcl cells within morphologically identifiable vacuolar structures. Receptor-mediated internalization of the mating pheromone alpha-factor occurred in chcl cells at 36-50% wild-type levels. The mutant cells were fully competent to respond to pheromone-induced cell-cycle arrest. These results argue that in yeast, clathrin may not play an essential role either in vacuolar protein sorting and delivery or in receptor-mediated endocytosis of alpha-factor. Alternative mechanisms ordinarily may execute these pathways, or be activated in clathrin-deficient cells.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Clathrin heavy chain-deficient mutants (chcl) of Saccharomyces cerevisiae are viable but exhibit compromised growth rates. To investigate the role of clathrin in intercompartmental protein transport, two pathways have been monitored in chcl cells: transport of newly synthesized vacuolar proteins to the vacuole and receptor-mediated uptake of mating pheromone. Newly synthesized precursors of the vacuolar protease carboxypeptidase Y (CPY) were converted to mature CPY with similar kinetics in mutant and wild-type cells. chcl cells did not aberrantly secrete CPY and immunolocalization techniques revealed most of the CPY in chcl cells within morphologically identifiable vacuolar structures. Receptor-mediated internalization of the mating pheromone alpha-factor occurred in chcl cells at 36-50% wild-type levels. The mutant cells were fully competent to respond to pheromone-induced cell-cycle arrest. These results argue that in yeast, clathrin may not play an essential role either in vacuolar protein sorting and delivery or in receptor-mediated endocytosis of alpha-factor. Alternative mechanisms ordinarily may execute these pathways, or be activated in clathrin-deficient cells.
2022
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2021
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Sahtoe, Danny D., Coscia, Adrian, Mustafaoglu, Nur, Miller, Lauren M., Olal, Daniel, Vulovic, Ivan, Yu, Ta-Yi, Goreshnik, Inna, Lin, Yu-Ru, Clark, Lars, Busch, Florian, Stewart, Lance, Wysocki, Vicki H., Ingber, Donald E., Abraham, Jonathan, Baker, David
Transferrin receptor targeting by de novo sheet extension Journal Article
In: Proceedings of the National Academy of Sciences, 2021.
@article{Sahtoe2021,
title = {Transferrin receptor targeting by de novo sheet extension},
author = {Sahtoe, Danny D. and Coscia, Adrian and Mustafaoglu, Nur and Miller, Lauren M. and Olal, Daniel and Vulovic, Ivan and Yu, Ta-Yi and Goreshnik, Inna and Lin, Yu-Ru and Clark, Lars and Busch, Florian and Stewart, Lance and Wysocki, Vicki H. and Ingber, Donald E. and Abraham, Jonathan and Baker, David},
url = {https://www.pnas.org/content/118/17/e2021569118, PNAS
},
doi = {10.1073/pnas.2021569118},
year = {2021},
date = {2021-04-27},
urldate = {2021-04-27},
journal = {Proceedings of the National Academy of Sciences},
abstract = {The de novo design of proteins that bind natural target proteins is useful for a variety of biomedical and biotechnological applications. We describe a design strategy to target proteins containing an exposed beta edge strand. We use the approach to design binders to the human transferrin receptor which shuttles back and forth across the blood{textendash}brain barrier. Such binders could be useful for the delivery of therapeutics into the brain.The de novo design of polar protein{textendash}protein interactions is challenging because of the thermodynamic cost of stripping water away from the polar groups. Here, we describe a general approach for designing proteins which complement exposed polar backbone groups at the edge of beta sheets with geometrically matched beta strands. We used this approach to computationally design small proteins that bind to an exposed beta sheet on the human transferrin receptor (hTfR), which shuttles interacting proteins across the blood{textendash}brain barrier (BBB), opening up avenues for drug delivery into the brain. We describe a design which binds hTfR with a 20 nM Kd, is hyperstable, and crosses an in vitro microfluidic organ-on-a-chip model of the human BBB. Our design approach provides a general strategy for creating binders to protein targets with exposed surface beta edge strands.Crystal structures have been deposited in the RCSB PDB with the accession nos. 6WRX, 6WRW, and 6WRV. Additional supporting data has been deposited in the online Zenodo repository (https://zenodo.org/record/4594115) (47). All other study data are included in the article and/or supporting information.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The de novo design of proteins that bind natural target proteins is useful for a variety of biomedical and biotechnological applications. We describe a design strategy to target proteins containing an exposed beta edge strand. We use the approach to design binders to the human transferrin receptor which shuttles back and forth across the blood{textendash}brain barrier. Such binders could be useful for the delivery of therapeutics into the brain.The de novo design of polar protein{textendash}protein interactions is challenging because of the thermodynamic cost of stripping water away from the polar groups. Here, we describe a general approach for designing proteins which complement exposed polar backbone groups at the edge of beta sheets with geometrically matched beta strands. We used this approach to computationally design small proteins that bind to an exposed beta sheet on the human transferrin receptor (hTfR), which shuttles interacting proteins across the blood{textendash}brain barrier (BBB), opening up avenues for drug delivery into the brain. We describe a design which binds hTfR with a 20 nM Kd, is hyperstable, and crosses an in vitro microfluidic organ-on-a-chip model of the human BBB. Our design approach provides a general strategy for creating binders to protein targets with exposed surface beta edge strands.Crystal structures have been deposited in the RCSB PDB with the accession nos. 6WRX, 6WRW, and 6WRV. Additional supporting data has been deposited in the online Zenodo repository (https://zenodo.org/record/4594115) (47). All other study data are included in the article and/or supporting information.
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2020
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2019
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2018
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2017–1998
ALL PAPERS
2017
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
Yang Hsia*, Jacob B. Bale*, Shane Gonen, Dan Shi, William Sheffler, Kimberly K. Fong, Nattermann, Chunfu Xu, Po-Ssu Huang, Rashmi Ravichandran, Sue Yi, Trisha N. Davis, Tamir Gonen, Neil P. King, David Baker
Design of a hyperstable 60-subunit protein icosahedron Journal Article
In: Nature, 2016.
@article{Hsia2016,
title = {Design of a hyperstable 60-subunit protein icosahedron},
author = { Yang Hsia* and Jacob B. Bale* and Shane Gonen and Dan Shi and William Sheffler and Kimberly K. Fong and Nattermann and Chunfu Xu and Po-Ssu Huang and Rashmi Ravichandran and Sue Yi and Trisha N. Davis and Tamir Gonen and Neil P. King and David Baker},
url = {https://www.bakerlab.org/wp-content/uploads/2016/06/Hsia_Nature_2016.pdf},
doi = {10.1038/nature18010},
year = {2016},
date = {2016-06-15},
journal = {Nature},
abstract = {The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/ exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/ exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.
2014
Erik Procko, Geoffrey Y Berguig, Betty W Shen, Yifan Song, Shani Frayo, Anthony J Convertine, Daciana Margineantu, Garrett Booth, Bruno E Correia, Yuanhua Cheng, William R Schief, David M Hockenbery, Oliver W Press, Barry L Stoddard, Patrick S Stayton, David Baker
A computationally designed inhibitor of an epstein-barr viral bcl-2 protein induces apoptosis in infected cells. Journal Article
In: Cell, vol. 157, pp. 1644-56, 2014, ISSN: 1097-4172.
@article{538,
title = {A computationally designed inhibitor of an epstein-barr viral bcl-2 protein induces apoptosis in infected cells.},
author = { Erik Procko and Geoffrey Y Berguig and Betty W Shen and Yifan Song and Shani Frayo and Anthony J Convertine and Daciana Margineantu and Garrett Booth and Bruno E Correia and Yuanhua Cheng and William R Schief and David M Hockenbery and Oliver W Press and Barry L Stoddard and Patrick S Stayton and David Baker},
url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Procko2014A.pdf},
doi = {10.1016/j.cell.2014.04.034},
issn = {1097-4172},
year = {2014},
date = {2014-06-01},
journal = {Cell},
volume = {157},
pages = {1644-56},
abstract = {Because apoptosis of infected cells can limit virus production and spread, some viruses have co-opted prosurvival genes from the host. This includes the Epstein-Barr virus (EBV) gene BHRF1, a homolog of human Bcl-2 proteins that block apoptosis and are associated with cancer. Computational design and experimental optimization were used to generate a novel protein called BINDI that binds BHRF1 with picomolar affinity. BINDI recognizes the hydrophobic cleft of BHRF1 in a manner similar to other Bcl-2 protein interactions but makes many additional contacts to achieve exceptional affinity and specificity. BINDI induces apoptosis in EBV-infected cancer lines, and when delivered with an antibody-targeted intracellular delivery carrier, BINDI suppressed tumor growth and extended survival in a xenograft disease model of EBV-positive human lymphoma. High-specificity-designed proteins that selectively kill target cells may provide an advantage over the toxic compounds used in current generation antibody-drug conjugates.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Because apoptosis of infected cells can limit virus production and spread, some viruses have co-opted prosurvival genes from the host. This includes the Epstein-Barr virus (EBV) gene BHRF1, a homolog of human Bcl-2 proteins that block apoptosis and are associated with cancer. Computational design and experimental optimization were used to generate a novel protein called BINDI that binds BHRF1 with picomolar affinity. BINDI recognizes the hydrophobic cleft of BHRF1 in a manner similar to other Bcl-2 protein interactions but makes many additional contacts to achieve exceptional affinity and specificity. BINDI induces apoptosis in EBV-infected cancer lines, and when delivered with an antibody-targeted intracellular delivery carrier, BINDI suppressed tumor growth and extended survival in a xenograft disease model of EBV-positive human lymphoma. High-specificity-designed proteins that selectively kill target cells may provide an advantage over the toxic compounds used in current generation antibody-drug conjugates.
2013
Sandrine Boissel, Jordan Jarjour, Alexander Astrakhan, Andrew Adey, Agn`es Gouble, Philippe Duchateau, Jay Shendure, Barry L Stoddard, Michael T Certo, David Baker, Andrew M Scharenberg
megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering. Journal Article
In: Nucleic acids research, 2013, ISSN: 1362-4962.
@article{516,
title = {megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering.},
author = { Sandrine Boissel and Jordan Jarjour and Alexander Astrakhan and Andrew Adey and Agn`es Gouble and Philippe Duchateau and Jay Shendure and Barry L Stoddard and Michael T Certo and David Baker and Andrew M Scharenberg},
url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Boissel_NAR_2013.pdf},
issn = {1362-4962},
year = {2013},
date = {2013-11-01},
journal = {Nucleic acids research},
abstract = {Rare-cleaving endonucleases have emerged as important tools for making targeted genome modifications. While multiple platforms are now available to generate reagents for research applications, each existing platform has significant limitations in one or more of three key properties necessary for therapeutic application: efficiency of cleavage at the desired target site, specificity of cleavage (i.e. rate of cleavage at textquoterightoff-targettextquoteright sites), and efficient/facile means for delivery to desired target cells. Here, we describe the development of a single-chain rare-cleaving nuclease architecture, which we designate textquoterightmegaTALtextquoteright, in which the DNA binding region of a transcription activator-like (TAL) effector is used to textquoterightaddresstextquoteright a site-specific meganuclease adjacent to a single desired genomic target site. This architecture allows the generation of extremely active and hyper-specific compact nucleases that are compatible with all current viral and nonviral cell delivery methods.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Rare-cleaving endonucleases have emerged as important tools for making targeted genome modifications. While multiple platforms are now available to generate reagents for research applications, each existing platform has significant limitations in one or more of three key properties necessary for therapeutic application: efficiency of cleavage at the desired target site, specificity of cleavage (i.e. rate of cleavage at textquoterightoff-targettextquoteright sites), and efficient/facile means for delivery to desired target cells. Here, we describe the development of a single-chain rare-cleaving nuclease architecture, which we designate textquoterightmegaTALtextquoteright, in which the DNA binding region of a transcription activator-like (TAL) effector is used to textquoterightaddresstextquoteright a site-specific meganuclease adjacent to a single desired genomic target site. This architecture allows the generation of extremely active and hyper-specific compact nucleases that are compatible with all current viral and nonviral cell delivery methods.
Julien R C Bergeron, Liam J Worrall, Nikolaos G Sgourakis, Frank DiMaio, Richard A Pfuetzner, Heather B Felise, Marija Vuckovic, Angel C Yu, Samuel I Miller, David Baker, Natalie C J Strynadka
A Refined Model of the Prototypical Salmonella SPI-1 T3SS Basal Body Reveals the Molecular Basis for Its Assembly Journal Article
In: PLoS pathogens, vol. 9, pp. e1003307, 2013, ISSN: 1553-7374.
@article{468,
title = {A Refined Model of the Prototypical Salmonella SPI-1 T3SS Basal Body Reveals the Molecular Basis for Its Assembly},
author = { Julien R C Bergeron and Liam J Worrall and Nikolaos G Sgourakis and Frank DiMaio and Richard A Pfuetzner and Heather B Felise and Marija Vuckovic and Angel C Yu and Samuel I Miller and David Baker and Natalie C J Strynadka},
url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Bergeron_ppat1003307_13T.pdf},
doi = {10.1371/journal.ppat.1003307},
issn = {1553-7374},
year = {2013},
date = {2013-04-01},
journal = {PLoS pathogens},
volume = {9},
pages = {e1003307},
abstract = {The T3SS injectisome is a syringe-shaped macromolecular assembly found in pathogenic Gram-negative bacteria that allows for the direct delivery of virulence effectors into host cells. It is composed of a "basal body", a lock-nut structure spanning both bacterial membranes, and a "needle" that protrudes away from the bacterial surface. A hollow channel spans throughout the apparatus, permitting the translocation of effector proteins from the bacterial cytosol to the host plasma membrane. The basal body is composed largely of three membrane-embedded proteins that form oligomerized concentric rings. Here, we report the crystal structures of three domains of the prototypical Salmonella SPI-1 basal body, and use a new approach incorporating symmetric flexible backbone docking and EM data to produce a model for their oligomeric assembly. The obtained models, validated by biochemical and in vivo assays, reveal the molecular details of the interactions driving basal body assembly, and notably demonstrate a conserved oligomerization mechanism.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The T3SS injectisome is a syringe-shaped macromolecular assembly found in pathogenic Gram-negative bacteria that allows for the direct delivery of virulence effectors into host cells. It is composed of a "basal body", a lock-nut structure spanning both bacterial membranes, and a "needle" that protrudes away from the bacterial surface. A hollow channel spans throughout the apparatus, permitting the translocation of effector proteins from the bacterial cytosol to the host plasma membrane. The basal body is composed largely of three membrane-embedded proteins that form oligomerized concentric rings. Here, we report the crystal structures of three domains of the prototypical Salmonella SPI-1 basal body, and use a new approach incorporating symmetric flexible backbone docking and EM data to produce a model for their oligomeric assembly. The obtained models, validated by biochemical and in vivo assays, reveal the molecular details of the interactions driving basal body assembly, and notably demonstrate a conserved oligomerization mechanism.
1988
G S Payne, D Baker, E van Tuinen, R Schekman
Protein transport to the vacuole and receptor-mediated endocytosis by clathrin heavy chain-deficient yeast Journal Article
In: The Journal of cell biology, vol. 106, pp. 1453-61, 1988, ISSN: 0021-9525.
@article{333,
title = {Protein transport to the vacuole and receptor-mediated endocytosis by clathrin heavy chain-deficient yeast},
author = { G S Payne and D Baker and E van Tuinen and R Schekman},
url = {https://www.bakerlab.org/wp-content/uploads/2016/06/payne88A.pdf},
issn = {0021-9525},
year = {1988},
date = {1988-05-01},
journal = {The Journal of cell biology},
volume = {106},
pages = {1453-61},
abstract = {Clathrin heavy chain-deficient mutants (chcl) of Saccharomyces cerevisiae are viable but exhibit compromised growth rates. To investigate the role of clathrin in intercompartmental protein transport, two pathways have been monitored in chcl cells: transport of newly synthesized vacuolar proteins to the vacuole and receptor-mediated uptake of mating pheromone. Newly synthesized precursors of the vacuolar protease carboxypeptidase Y (CPY) were converted to mature CPY with similar kinetics in mutant and wild-type cells. chcl cells did not aberrantly secrete CPY and immunolocalization techniques revealed most of the CPY in chcl cells within morphologically identifiable vacuolar structures. Receptor-mediated internalization of the mating pheromone alpha-factor occurred in chcl cells at 36-50% wild-type levels. The mutant cells were fully competent to respond to pheromone-induced cell-cycle arrest. These results argue that in yeast, clathrin may not play an essential role either in vacuolar protein sorting and delivery or in receptor-mediated endocytosis of alpha-factor. Alternative mechanisms ordinarily may execute these pathways, or be activated in clathrin-deficient cells.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Clathrin heavy chain-deficient mutants (chcl) of Saccharomyces cerevisiae are viable but exhibit compromised growth rates. To investigate the role of clathrin in intercompartmental protein transport, two pathways have been monitored in chcl cells: transport of newly synthesized vacuolar proteins to the vacuole and receptor-mediated uptake of mating pheromone. Newly synthesized precursors of the vacuolar protease carboxypeptidase Y (CPY) were converted to mature CPY with similar kinetics in mutant and wild-type cells. chcl cells did not aberrantly secrete CPY and immunolocalization techniques revealed most of the CPY in chcl cells within morphologically identifiable vacuolar structures. Receptor-mediated internalization of the mating pheromone alpha-factor occurred in chcl cells at 36-50% wild-type levels. The mutant cells were fully competent to respond to pheromone-induced cell-cycle arrest. These results argue that in yeast, clathrin may not play an essential role either in vacuolar protein sorting and delivery or in receptor-mediated endocytosis of alpha-factor. Alternative mechanisms ordinarily may execute these pathways, or be activated in clathrin-deficient cells.