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2015 |
Vittal, Vinayak; Shi, Lei; Wenzel, Dawn M; Scaglione, Matthew K; Duncan, Emily D; Basrur, Venkatesha; Elenitoba-Johnson, Kojo S J; Baker, David; Paulson, Henry L; Brzovic, Peter S; Klevit, Rachel E Intrinsic disorder drives N-terminal ubiquitination by Ube2w Journal Article Nature Chemical Biology, 11 , pp. 83-9, 2015, ISSN: 1552-4469. @article{610, title = {Intrinsic disorder drives N-terminal ubiquitination by Ube2w}, author = { Vinayak Vittal and Lei Shi and Dawn M Wenzel and K Matthew Scaglione and Emily D Duncan and Venkatesha Basrur and Kojo S J Elenitoba-Johnson and David Baker and Henry L Paulson and Peter S Brzovic and Rachel E Klevit}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/intrinsicdisorderdrives_Baker2015.pdf}, doi = {10.1038/nchembio.1700}, issn = {1552-4469}, year = {2015}, date = {2015-01-01}, journal = {Nature Chemical Biology}, volume = {11}, pages = {83-9}, abstract = {Ubiquitination of the αN-terminus of protein substrates has been reported sporadically since the early 1980s. However, the identity of an enzyme responsible for this unique ubiquitin (Ub) modification has only recently been elucidated. We show the Ub-conjugating enzyme (E2) Ube2w uses a unique mechanism to facilitate the specific ubiquitination of the α-amino group of its substrates that involves recognition of backbone atoms of intrinsically disordered N termini. We present the NMR-based solution ensemble of full-length Ube2w that reveals a structural architecture unlike that of any other E2 in which its C terminus is partly disordered and flexible to accommodate variable substrate N termini. Flexibility of the substrate is critical for recognition by Ube2w, and either point mutations in or the removal of the flexible C terminus of Ube2w inhibits substrate binding and modification. Mechanistic insights reported here provide guiding principles for future efforts to define the N-terminal ubiquitome in cells.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Ubiquitination of the αN-terminus of protein substrates has been reported sporadically since the early 1980s. However, the identity of an enzyme responsible for this unique ubiquitin (Ub) modification has only recently been elucidated. We show the Ub-conjugating enzyme (E2) Ube2w uses a unique mechanism to facilitate the specific ubiquitination of the α-amino group of its substrates that involves recognition of backbone atoms of intrinsically disordered N termini. We present the NMR-based solution ensemble of full-length Ube2w that reveals a structural architecture unlike that of any other E2 in which its C terminus is partly disordered and flexible to accommodate variable substrate N termini. Flexibility of the substrate is critical for recognition by Ube2w, and either point mutations in or the removal of the flexible C terminus of Ube2w inhibits substrate binding and modification. Mechanistic insights reported here provide guiding principles for future efforts to define the N-terminal ubiquitome in cells. |
Park, Keunwan; Shen, Betty W; Parmeggiani, Fabio; Huang, Po-Ssu; Stoddard, Barry L; Baker, David Control of repeat-protein curvature by computational protein design. Journal Article Nature structural & molecular biology, 2015, ISSN: 1545-9985. @article{557, title = {Control of repeat-protein curvature by computational protein design.}, author = { Keunwan Park and Betty W Shen and Fabio Parmeggiani and Po-Ssu Huang and Barry L Stoddard and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Park_2015.pdf}, doi = {10.1038/nsmb.2938}, issn = {1545-9985}, year = {2015}, date = {2015-01-01}, journal = {Nature structural & molecular biology}, abstract = {Shape complementarity is an important component of molecular recognition, and the ability to precisely adjust the shape of a binding scaffold to match a target of interest would greatly facilitate the creation of high-affinity protein reagents and therapeutics. Here we describe a general approach to control the shape of the binding surface on repeat-protein scaffolds and apply it to leucine-rich-repeat proteins. First, self-compatible building-block modules are designed that, when polymerized, generate surfaces with unique but constant curvatures. Second, a set of junction modules that connect the different building blocks are designed. Finally, new proteins with custom-designed shapes are generated by appropriately combining building-block and junction modules. Crystal structures of the designs illustrate the power of the approach in controlling repeat-protein curvature.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Shape complementarity is an important component of molecular recognition, and the ability to precisely adjust the shape of a binding scaffold to match a target of interest would greatly facilitate the creation of high-affinity protein reagents and therapeutics. Here we describe a general approach to control the shape of the binding surface on repeat-protein scaffolds and apply it to leucine-rich-repeat proteins. First, self-compatible building-block modules are designed that, when polymerized, generate surfaces with unique but constant curvatures. Second, a set of junction modules that connect the different building blocks are designed. Finally, new proteins with custom-designed shapes are generated by appropriately combining building-block and junction modules. Crystal structures of the designs illustrate the power of the approach in controlling repeat-protein curvature. |
Bergeron, Julien R C; Worrall, Liam J; De, Soumya; Sgourakis, Nikolaos G; Cheung, Adrienne H; Lameignere, Emilie; Okon, Mark; Wasney, Gregory A; Baker, David; McIntosh, Lawrence P; Strynadka, Natalie C J The modular structure of the inner-membrane ring component PrgK facilitates assembly of the type III secretion system basal body Journal Article Structure (London, England : 1993), 23 , pp. 161-72, 2015, ISSN: 1878-4186. @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} } 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. |
2014 |
Thyme, Summer B; Song, Yifan; Brunette, Tj; Szeto, Mindy D; Kusak, Lara; Bradley, Philip; Baker, David Massively parallel determination and modeling of endonuclease substrate specificity Journal Article Nucleic Acids Research, 42 , pp. 13839-52, 2014, ISSN: 1362-4962. @article{606, title = {Massively parallel determination and modeling of endonuclease substrate specificity}, author = { Summer B Thyme and Yifan Song and Tj Brunette and Mindy D Szeto and Lara Kusak and Philip Bradley and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/massivelyparallel_Baker2013.pdf}, doi = {10.1093/nar/gku1096}, issn = {1362-4962}, year = {2014}, date = {2014-12-01}, journal = {Nucleic Acids Research}, volume = {42}, pages = {13839-52}, abstract = {We describe the identification and characterization of novel homing endonucleases using genome database mining to identify putative target sites, followed by high throughput activity screening in a bacterial selection system. We characterized the substrate specificity and kinetics of these endonucleases by monitoring DNA cleavage events with deep sequencing. The endonuclease specificities revealed by these experiments can be partially recapitulated using 3D structure-based computational models. Analysis of these models together with genome sequence data provide insights into how alternative endonuclease specificities were generated during natural evolution.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We describe the identification and characterization of novel homing endonucleases using genome database mining to identify putative target sites, followed by high throughput activity screening in a bacterial selection system. We characterized the substrate specificity and kinetics of these endonucleases by monitoring DNA cleavage events with deep sequencing. The endonuclease specificities revealed by these experiments can be partially recapitulated using 3D structure-based computational models. Analysis of these models together with genome sequence data provide insights into how alternative endonuclease specificities were generated during natural evolution. |
Parmeggiani, Fabio; Huang, Po-Ssu; Vorobiev, Sergey; Xiao, Rong; Park, Keunwan; Caprari, Silvia; Su, Min; Seetharaman, Jayaraman; Mao, Lei; Janjua, Haleema; Montelione, Gaetano T; Hunt, John; Baker, David A General Computational Approach for Repeat Protein Design. Journal Article Journal of molecular biology, 2014, ISSN: 1089-8638. @article{555, title = {A General Computational Approach for Repeat Protein Design.}, author = { Fabio Parmeggiani and Po-Ssu Huang and Sergey Vorobiev and Rong Xiao and Keunwan Park and Silvia Caprari and Min Su and Jayaraman Seetharaman and Lei Mao and Haleema Janjua and Gaetano T Montelione and John Hunt and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Parmeggiani-2014.pdf}, doi = {10.1016/j.jmb.2014.11.005}, issn = {1089-8638}, year = {2014}, date = {2014-11-01}, journal = {Journal of molecular biology}, abstract = {Repeat proteins have considerable potential for use as modular binding reagents or biomaterials in biomedical and nanotechnology applications. Here we describe a general computational method for building idealized repeats that integrates available family sequences and structural information with Rosetta de novo protein design calculations. Idealized designs from six different repeat families were generated and experimentally characterized; 80% of the proteins were expressed and soluble and more than 40% were folded and monomeric with high thermal stability. Crystal structures determined for members of three families are within 1r A root-mean-square deviation to the design models. The method provides a general approach for fast and reliable generation of stable modular repeat protein scaffolds.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Repeat proteins have considerable potential for use as modular binding reagents or biomaterials in biomedical and nanotechnology applications. Here we describe a general computational method for building idealized repeats that integrates available family sequences and structural information with Rosetta de novo protein design calculations. Idealized designs from six different repeat families were generated and experimentally characterized; 80% of the proteins were expressed and soluble and more than 40% were folded and monomeric with high thermal stability. Crystal structures determined for members of three families are within 1r A root-mean-square deviation to the design models. The method provides a general approach for fast and reliable generation of stable modular repeat protein scaffolds. |
Liu, Daniel S; Niv'on, Lucas G; Richter, Florian; Goldman, Peter J; Deerinck, Thomas J; Yao, Jennifer Z; Richardson, Douglas; Phipps, William S; Ye, Anne Z; Ellisman, Mark H; Drennan, Catherine L; Baker, David; Ting, Alice Y Computational design of a red fluorophore ligase for site-specific protein labeling in living cells. Journal Article Proceedings of the National Academy of Sciences of the United States of America, 111 , pp. E4551-9, 2014, ISSN: 1091-6490. @article{619, title = {Computational design of a red fluorophore ligase for site-specific protein labeling in living cells.}, author = { Daniel S Liu and Lucas G Niv'on and Florian Richter and Peter J Goldman and Thomas J Deerinck and Jennifer Z Yao and Douglas Richardson and William S Phipps and Anne Z Ye and Mark H Ellisman and Catherine L Drennan and David Baker and Alice Y Ting}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Liu_computational_PNAS_2014.pdf}, doi = {10.1073/pnas.1404736111}, issn = {1091-6490}, year = {2014}, date = {2014-10-01}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {111}, pages = {E4551-9}, abstract = {Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies. |
Huang, P; Oberdorfer, G; Xu, C; Pei, X Y; Nannenga, B L; Rogers, J M; DiMaio, F; Gonen, T; Luisi, B; Baker, D High thermodynamic stability of parametrically designed helical bundles Journal Article Science, 346 , pp. 481-85, 2014. @article{552, title = {High thermodynamic stability of parametrically designed helical bundles}, author = { P. Huang and G. Oberdorfer and C. Xu and X.Y. Pei and B.L. Nannenga and J.M. Rogers and F. DiMaio and T. Gonen and B. Luisi and D Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Huang_2014.pdf}, doi = {10.1126/science.1257481}, year = {2014}, date = {2014-10-01}, journal = {Science}, volume = {346}, pages = {481-85}, chapter = {481}, abstract = {We describe a procedure for designing proteins with backbones produced by varying the parameters in the Crick coiled coil–generating equations. Combinatorial design calculations identify low-energy sequences for alternative helix supercoil arrangements, and the helices in the lowest-energy arrangements are connected by loop building. We design an antiparallel monomeric untwisted three-helix bundle with 80-residue helices, an antiparallel monomeric right-handed four-helix bundle, and a pentameric parallel left-handed five-helix bundle. The designed proteins are extremely stable (extrapolated ΔGfold > 60 kilocalories per mole), and their crystal structures are close to those of the design models with nearly identical core packing between the helices. The approach enables the custom design of hyperstable proteins with fine-tuned geometries for a wide range of applications.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We describe a procedure for designing proteins with backbones produced by varying the parameters in the Crick coiled coil–generating equations. Combinatorial design calculations identify low-energy sequences for alternative helix supercoil arrangements, and the helices in the lowest-energy arrangements are connected by loop building. We design an antiparallel monomeric untwisted three-helix bundle with 80-residue helices, an antiparallel monomeric right-handed four-helix bundle, and a pentameric parallel left-handed five-helix bundle. The designed proteins are extremely stable (extrapolated ΔGfold > 60 kilocalories per mole), and their crystal structures are close to those of the design models with nearly identical core packing between the helices. The approach enables the custom design of hyperstable proteins with fine-tuned geometries for a wide range of applications. |
Liu, Yu; Zhang, Xin; Tan, Yun Lei; Bhabha, Gira; Ekiert, Damian C; Kipnis, Yakov; Bjelic, Sinisa; Baker, David; Kelly, Jeffery W De novo-designed enzymes as small-molecule-regulated fluorescence imaging tags and fluorescent reporters. Journal Article Journal of the American Chemical Society, 136 , pp. 13102-5, 2014, ISSN: 1520-5126. @article{621, title = {De novo-designed enzymes as small-molecule-regulated fluorescence imaging tags and fluorescent reporters.}, author = { Yu Liu and Xin Zhang and Yun Lei Tan and Gira Bhabha and Damian C Ekiert and Yakov Kipnis and Sinisa Bjelic and David Baker and Jeffery W Kelly}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Liu_JACS_2014.pdf}, doi = {10.1021/ja5056356}, issn = {1520-5126}, year = {2014}, date = {2014-09-01}, journal = {Journal of the American Chemical Society}, volume = {136}, pages = {13102-5}, abstract = {Enzyme-based tags attached to a protein-of-interest (POI) that react with a small molecule, rendering the conjugate fluorescent, are very useful for studying the POI in living cells. These tags are typically based on endogenous enzymes, so protein engineering is required to ensure that the small-molecule probe does not react with the endogenous enzyme in the cell of interest. Here we demonstrate that de novo-designed enzymes can be used as tags to attach to POIs. The inherent bioorthogonality of the de novo-designed enzyme-small-molecule probe reaction circumvents the need for protein engineering, since these enzyme activities are not present in living organisms. Herein, we transform a family of de novo-designed retroaldolases into variable-molecular-weight tags exhibiting fluorescence imaging, reporter, and electrophoresis applications that are regulated by tailored, reactive small-molecule fluorophores.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Enzyme-based tags attached to a protein-of-interest (POI) that react with a small molecule, rendering the conjugate fluorescent, are very useful for studying the POI in living cells. These tags are typically based on endogenous enzymes, so protein engineering is required to ensure that the small-molecule probe does not react with the endogenous enzyme in the cell of interest. Here we demonstrate that de novo-designed enzymes can be used as tags to attach to POIs. The inherent bioorthogonality of the de novo-designed enzyme-small-molecule probe reaction circumvents the need for protein engineering, since these enzyme activities are not present in living organisms. Herein, we transform a family of de novo-designed retroaldolases into variable-molecular-weight tags exhibiting fluorescence imaging, reporter, and electrophoresis applications that are regulated by tailored, reactive small-molecule fluorophores. |
Khoury, George A; Liwo, Adam; Khatib, Firas; Zhou, Hongyi; Chopra, Gaurav; Bacardit, Jaume; Bortot, Leandro O; Faccioli, Rodrigo A; Deng, Xin; He, Yi; Krupa, Pawel; Li, Jilong; Mozolewska, Magdalena A; Sieradzan, Adam K; Smadbeck, James; Wirecki, Tomasz; Cooper, Seth; Flatten, Jeff; Xu, Kefan; Baker, David; Cheng, Jianlin; Delbem, Alexandre C B; Floudas, Christodoulos A; Keasar, Chen; Levitt, Michael; Popovi'c, Zoran; Scheraga, Harold A; Skolnick, Jeffrey; Crivelli, Silvia N WeFold: a coopetition for protein structure prediction. Journal Article Proteins, 82 , pp. 1850-68, 2014, ISSN: 1097-0134. @article{625, title = {WeFold: a coopetition for protein structure prediction.}, author = { George A Khoury and Adam Liwo and Firas Khatib and Hongyi Zhou and Gaurav Chopra and Jaume Bacardit and Leandro O Bortot and Rodrigo A Faccioli and Xin Deng and Yi He and Pawel Krupa and Jilong Li and Magdalena A Mozolewska and Adam K Sieradzan and James Smadbeck and Tomasz Wirecki and Seth Cooper and Jeff Flatten and Kefan Xu and David Baker and Jianlin Cheng and Alexandre C B Delbem and Christodoulos A Floudas and Chen Keasar and Michael Levitt and Zoran Popovi'c and Harold A Scheraga and Jeffrey Skolnick and Silvia N Crivelli}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Khoury_Proteins_2014.pdf}, doi = {10.1002/prot.24538}, issn = {1097-0134}, year = {2014}, date = {2014-09-01}, journal = {Proteins}, volume = {82}, pages = {1850-68}, abstract = {The protein structure prediction problem continues to elude scientists. Despite the introduction of many methods, only modest gains were made over the last decade for certain classes of prediction targets. To address this challenge, a social-media based worldwide collaborative effort, named WeFold, was undertaken by 13 labs. During the collaboration, the laboratories were simultaneously competing with each other. Here, we present the first attempt at "coopetition" in scientific research applied to the protein structure prediction and refinement problems. The coopetition was possible by allowing the participating labs to contribute different components of their protein structure prediction pipelines and create new hybrid pipelines that they tested during CASP10. This manuscript describes both successes and areas needing improvement as identified throughout the first WeFold experiment and discusses the efforts that are underway to advance this initiative. A footprint of all contributions and structures are publicly accessible at http://www.wefold.org.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The protein structure prediction problem continues to elude scientists. Despite the introduction of many methods, only modest gains were made over the last decade for certain classes of prediction targets. To address this challenge, a social-media based worldwide collaborative effort, named WeFold, was undertaken by 13 labs. During the collaboration, the laboratories were simultaneously competing with each other. Here, we present the first attempt at "coopetition" in scientific research applied to the protein structure prediction and refinement problems. The coopetition was possible by allowing the participating labs to contribute different components of their protein structure prediction pipelines and create new hybrid pipelines that they tested during CASP10. This manuscript describes both successes and areas needing improvement as identified throughout the first WeFold experiment and discusses the efforts that are underway to advance this initiative. A footprint of all contributions and structures are publicly accessible at http://www.wefold.org. |
Griss, Rudolf; Schena, Alberto; Reymond, Luc; Patiny, Luc; Werner, Dominique; Tinberg, Christine E; Baker, David; Johnsson, Kai Bioluminescent sensor proteins for point-of-care therapeutic drug monitoring Journal Article Nature chemical biology, 10 , pp. 598-603, 2014, ISSN: 1552-4469. @article{539, title = {Bioluminescent sensor proteins for point-of-care therapeutic drug monitoring}, author = { Rudolf Griss and Alberto Schena and Luc Reymond and Luc Patiny and Dominique Werner and Christine E Tinberg and David Baker and Kai Johnsson}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Griss_2014A.pdf}, doi = {10.1038/nchembio.1554}, issn = {1552-4469}, year = {2014}, date = {2014-07-01}, journal = {Nature chemical biology}, volume = {10}, pages = {598-603}, abstract = {For many drugs, finding the balance between efficacy and toxicity requires monitoring their concentrations in the patienttextquoterights blood. Quantifying drug levels at the bedside or at home would have advantages in terms of therapeutic outcome and convenience, but current techniques require the setting of a diagnostic laboratory. We have developed semisynthetic bioluminescent sensors that permit precise measurements of drug concentrations in patient samples by spotting minimal volumes on paper and recording the signal using a simple point-and-shoot camera. Our sensors have a modular design consisting of a protein-based and a synthetic part and can be engineered to selectively recognize a wide range of drugs, including immunosuppressants, antiepileptics, anticancer agents and antiarrhythmics. This low-cost point-of-care method could make therapies safer, increase the convenience of doctors and patients and make therapeutic drug monitoring available in regions with poor infrastructure.}, keywords = {}, pubstate = {published}, tppubtype = {article} } For many drugs, finding the balance between efficacy and toxicity requires monitoring their concentrations in the patienttextquoterights blood. Quantifying drug levels at the bedside or at home would have advantages in terms of therapeutic outcome and convenience, but current techniques require the setting of a diagnostic laboratory. We have developed semisynthetic bioluminescent sensors that permit precise measurements of drug concentrations in patient samples by spotting minimal volumes on paper and recording the signal using a simple point-and-shoot camera. Our sensors have a modular design consisting of a protein-based and a synthetic part and can be engineered to selectively recognize a wide range of drugs, including immunosuppressants, antiepileptics, anticancer agents and antiarrhythmics. This low-cost point-of-care method could make therapies safer, increase the convenience of doctors and patients and make therapeutic drug monitoring available in regions with poor infrastructure. |
Preiswerk, Nathalie; Beck, Tobias; Schulz, Jessica D; Milovn'ik, Peter; Mayer, Clemens; Siegel, Justin B; Baker, David; Hilvert, Donald Impact of scaffold rigidity on the design and evolution of an artificial Diels-Alderase. Journal Article Proceedings of the National Academy of Sciences of the United States of America, 111 , pp. 8013-8, 2014, ISSN: 1091-6490. @article{623, title = {Impact of scaffold rigidity on the design and evolution of an artificial Diels-Alderase.}, author = { Nathalie Preiswerk and Tobias Beck and Jessica D Schulz and Peter Milovn'ik and Clemens Mayer and Justin B Siegel and David Baker and Donald Hilvert}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Preiswerk_PNAS_2014.pdf}, doi = {10.1073/pnas.1401073111}, issn = {1091-6490}, year = {2014}, date = {2014-06-01}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {111}, pages = {8013-8}, abstract = {By combining targeted mutagenesis, computational refinement, and directed evolution, a modestly active, computationally designed Diels-Alderase was converted into the most proficient biocatalyst for [4+2] cycloadditions known. The high stereoselectivity and minimal product inhibition of the evolved enzyme enabled preparative scale synthesis of a single product diastereomer. X-ray crystallography of the enzyme-product complex shows that the molecular changes introduced over the course of optimization, including addition of a lid structure, gradually reshaped the pocket for more effective substrate preorganization and transition state stabilization. The good overall agreement between the experimental structure and the original design model with respect to the orientations of both the bound product and the catalytic side chains contrasts with other computationally designed enzymes. Because design accuracy appears to correlate with scaffold rigidity, improved control over backbone conformation will likely be the key to future efforts to design more efficient enzymes for diverse chemical reactions.}, keywords = {}, pubstate = {published}, tppubtype = {article} } By combining targeted mutagenesis, computational refinement, and directed evolution, a modestly active, computationally designed Diels-Alderase was converted into the most proficient biocatalyst for [4+2] cycloadditions known. The high stereoselectivity and minimal product inhibition of the evolved enzyme enabled preparative scale synthesis of a single product diastereomer. X-ray crystallography of the enzyme-product complex shows that the molecular changes introduced over the course of optimization, including addition of a lid structure, gradually reshaped the pocket for more effective substrate preorganization and transition state stabilization. The good overall agreement between the experimental structure and the original design model with respect to the orientations of both the bound product and the catalytic side chains contrasts with other computationally designed enzymes. Because design accuracy appears to correlate with scaffold rigidity, improved control over backbone conformation will likely be the key to future efforts to design more efficient enzymes for diverse chemical reactions. |
Mazor, Ronit; Eberle, Jaime A; Hu, Xiaobo; Vassall, Aaron N; Onda, Masanori; Beers, Richard; Lee, Elizabeth C; Kreitman, Robert J; Lee, Byungkook; Baker, David; King, Chris; Hassan, Raffit; Benhar, Itai; Pastan, Ira Recombinant immunotoxin for cancer treatment with low immunogenicity by identification and silencing of human T-cell epitopes. Journal Article Proceedings of the National Academy of Sciences of the United States of America, 111 , pp. 8571-6, 2014, ISSN: 1091-6490. @article{624, title = {Recombinant immunotoxin for cancer treatment with low immunogenicity by identification and silencing of human T-cell epitopes.}, author = { Ronit Mazor and Jaime A Eberle and Xiaobo Hu and Aaron N Vassall and Masanori Onda and Richard Beers and Elizabeth C Lee and Robert J Kreitman and Byungkook Lee and David Baker and Chris King and Raffit Hassan and Itai Benhar and Ira Pastan}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Mazor_PNAS_2014.pdf}, doi = {10.1073/pnas.1405153111}, issn = {1091-6490}, year = {2014}, date = {2014-06-01}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {111}, pages = {8571-6}, abstract = {Nonhuman proteins have valuable therapeutic properties, but their efficacy is limited by neutralizing antibodies. Recombinant immunotoxins (RITs) are potent anticancer agents that have produced many complete remissions in leukemia, but immunogenicity limits the number of doses that can be given to patients with normal immune systems. Using human cells, we identified eight helper T-cell epitopes in PE38, a portion of the bacterial protein Pseudomonas exotoxin A which consists of the toxin moiety of the RIT, and used this information to make LMB-T18 in which three epitopes were deleted and five others diminished by point mutations in key residues. LMB-T18 has high cytotoxic and antitumor activity and is very resistant to thermal denaturation. The new immunotoxin has a 93% decrease in T-cell epitopes and should have improved efficacy in patients because more treatment cycles can be given. Furthermore, the deimmunized toxin can be used to make RITs targeting other antigens, and the approach we describe can be used to deimmunize other therapeutically useful nonhuman proteins.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Nonhuman proteins have valuable therapeutic properties, but their efficacy is limited by neutralizing antibodies. Recombinant immunotoxins (RITs) are potent anticancer agents that have produced many complete remissions in leukemia, but immunogenicity limits the number of doses that can be given to patients with normal immune systems. Using human cells, we identified eight helper T-cell epitopes in PE38, a portion of the bacterial protein Pseudomonas exotoxin A which consists of the toxin moiety of the RIT, and used this information to make LMB-T18 in which three epitopes were deleted and five others diminished by point mutations in key residues. LMB-T18 has high cytotoxic and antitumor activity and is very resistant to thermal denaturation. The new immunotoxin has a 93% decrease in T-cell epitopes and should have improved efficacy in patients because more treatment cycles can be given. Furthermore, the deimmunized toxin can be used to make RITs targeting other antigens, and the approach we describe can be used to deimmunize other therapeutically useful nonhuman proteins. |
Magad'an, Javier G; Altman, Meghan O; Ince, William L; Hickman, Heather D; Stevens, James; Chevalier, Aaron; Baker, David; Wilson, Patrick C; Ahmed, Rafi; Bennink, Jack R; Yewdell, Jonathan W Biogenesis of Influenza A Virus Hemagglutinin Cross-Protective Stem Epitopes. Journal Article PLoS pathogens, 10 , pp. e1004204, 2014, ISSN: 1553-7374. @article{537, title = {Biogenesis of Influenza A Virus Hemagglutinin Cross-Protective Stem Epitopes.}, author = { Javier G Magad'an and Meghan O Altman and William L Ince and Heather D Hickman and James Stevens and Aaron Chevalier and David Baker and Patrick C Wilson and Rafi Ahmed and Jack R Bennink and Jonathan W Yewdell}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Magadan_2014A.pdf}, doi = {10.1371/journal.ppat.1004204}, issn = {1553-7374}, year = {2014}, date = {2014-06-01}, journal = {PLoS pathogens}, volume = {10}, pages = {e1004204}, abstract = {Antigenic variation in the globular domain of influenza A virus (IAV) hemagglutinin (HA) precludes effective immunity to this major human pathogen. Although the HA stem is highly conserved between influenza virus strains, HA stem-reactive antibodies (StRAbs) were long considered biologically inert. It is now clear, however, that StRAbs reduce viral replication in animal models and protect against pathogenicity and death, supporting the potential of HA stem-based immunogens as drift-resistant vaccines. Optimally designing StRAb-inducing immunogens and understanding StRAb effector functions require thorough comprehension of HA stem structure and antigenicity. Here, we study the biogenesis of HA stem epitopes recognized in cells infected with various drifted IAV H1N1 strains using mouse and human StRAbs. Using a novel immunofluorescence (IF)-based assay, we find that human StRAbs bind monomeric HA in the endoplasmic reticulum (ER) and trimerized HA in the Golgi complex (GC) with similar high avidity, potentially good news for producing effective monomeric HA stem immunogens. Though HA stem epitopes are nestled among several N-linked oligosaccharides, glycosylation is not required for full antigenicity. Rather, as N-linked glycans increase in size during intracellular transport of HA through the GC, StRAb binding becomes temperature-sensitive, binding poorly to HA at 4textdegreeC and well at 37textdegreeC. A de novo designed, 65-residue protein binds the mature HA stem independently of temperature, consistent with a lack of N-linked oligosaccharide steric hindrance due to its small size. Likewise, StRAbs bind recombinant HA carrying simple N-linked glycans in a temperature-independent manner. Chemical cross-linking experiments show that N-linked oligosaccharides likely influence StRAb binding by direct local effects rather than by globally modifying the conformational flexibility of HA. Our findings indicate that StRAb binding to HA is precarious, raising the possibility that sufficient immune pressure on the HA stem region could select for viral escape mutants with increased steric hindrance from N-linked glycans.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Antigenic variation in the globular domain of influenza A virus (IAV) hemagglutinin (HA) precludes effective immunity to this major human pathogen. Although the HA stem is highly conserved between influenza virus strains, HA stem-reactive antibodies (StRAbs) were long considered biologically inert. It is now clear, however, that StRAbs reduce viral replication in animal models and protect against pathogenicity and death, supporting the potential of HA stem-based immunogens as drift-resistant vaccines. Optimally designing StRAb-inducing immunogens and understanding StRAb effector functions require thorough comprehension of HA stem structure and antigenicity. Here, we study the biogenesis of HA stem epitopes recognized in cells infected with various drifted IAV H1N1 strains using mouse and human StRAbs. Using a novel immunofluorescence (IF)-based assay, we find that human StRAbs bind monomeric HA in the endoplasmic reticulum (ER) and trimerized HA in the Golgi complex (GC) with similar high avidity, potentially good news for producing effective monomeric HA stem immunogens. Though HA stem epitopes are nestled among several N-linked oligosaccharides, glycosylation is not required for full antigenicity. Rather, as N-linked glycans increase in size during intracellular transport of HA through the GC, StRAb binding becomes temperature-sensitive, binding poorly to HA at 4textdegreeC and well at 37textdegreeC. A de novo designed, 65-residue protein binds the mature HA stem independently of temperature, consistent with a lack of N-linked oligosaccharide steric hindrance due to its small size. Likewise, StRAbs bind recombinant HA carrying simple N-linked glycans in a temperature-independent manner. Chemical cross-linking experiments show that N-linked oligosaccharides likely influence StRAb binding by direct local effects rather than by globally modifying the conformational flexibility of HA. Our findings indicate that StRAb binding to HA is precarious, raising the possibility that sufficient immune pressure on the HA stem region could select for viral escape mutants with increased steric hindrance from N-linked glycans. |
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 Cell, 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. |
Chen, Kuang-Yui M; Sun, Jiaming; Salvo, Jason S; Baker, David; Barth, Patrick High-resolution modeling of transmembrane helical protein structures from distant homologues. Journal Article PLoS computational biology, 10 , pp. e1003636, 2014, ISSN: 1553-7358. @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} } 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. |
King, Neil P; Bale, Jacob B; Sheffler, William; McNamara, Dan E; Gonen, Shane; Gonen, Tamir; Yeates, Todd O; Baker, David Accurate design of co-assembling multi-component protein nanomaterials. Journal Article Nature, 2014, ISSN: 1476-4687. @article{534, title = {Accurate design of co-assembling multi-component protein nanomaterials.}, author = { Neil P. King and Jacob B Bale and William Sheffler and Dan E McNamara and Shane Gonen and Tamir Gonen and Todd O. Yeates and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/King_Nature2014A.pdf}, doi = {10.1038/nature13404}, issn = {1476-4687}, year = {2014}, date = {2014-05-01}, journal = {Nature}, abstract = {The self-assembly of proteins into highly ordered nanoscale architectures is a hallmark of biological systems. The sophisticated functions of these molecular machines have inspired the development of methods to engineer self-assembling protein nanostructures; however, the design of multi-component protein nanomaterials with high accuracy remains an outstanding challenge. Here we report a computational method for designing protein nanomaterials in which multiple copies of two distinct subunits co-assemble into a specific architecture. We use the method to design five 24-subunit cage-like protein nanomaterials in two distinct symmetric architectures and experimentally demonstrate that their structures are in close agreement with the computational design models. The accuracy of the method and the number and variety of two-component materials that it makes accessible suggest a route to the construction of functional protein nanomaterials tailored to specific applications.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The self-assembly of proteins into highly ordered nanoscale architectures is a hallmark of biological systems. The sophisticated functions of these molecular machines have inspired the development of methods to engineer self-assembling protein nanostructures; however, the design of multi-component protein nanomaterials with high accuracy remains an outstanding challenge. Here we report a computational method for designing protein nanomaterials in which multiple copies of two distinct subunits co-assemble into a specific architecture. We use the method to design five 24-subunit cage-like protein nanomaterials in two distinct symmetric architectures and experimentally demonstrate that their structures are in close agreement with the computational design models. The accuracy of the method and the number and variety of two-component materials that it makes accessible suggest a route to the construction of functional protein nanomaterials tailored to specific applications. |
Alushin, Gregory M; Lander, Gabriel C; Kellogg, Elizabeth H; Zhang, Rui; Baker, David; Nogales, Eva High-resolution microtubule structures reveal the structural transitions in αβ-tubulin upon GTP hydrolysis. Journal Article Cell, 157 , pp. 1117-29, 2014, ISSN: 1097-4172. @article{541, title = {High-resolution microtubule structures reveal the structural transitions in αβ-tubulin upon GTP hydrolysis.}, author = { Gregory M Alushin and Gabriel C Lander and Elizabeth H Kellogg and Rui Zhang and David Baker and Eva Nogales}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Alushin_2014A.pdf}, doi = {10.1016/j.cell.2014.03.053}, issn = {1097-4172}, year = {2014}, date = {2014-05-01}, journal = {Cell}, volume = {157}, pages = {1117-29}, abstract = {Dynamic instability, the stochastic switching between growth and shrinkage, is essential for microtubule function. This behavior is driven by GTP hydrolysis in the microtubule lattice and is inhibited by anticancer agents like Taxol. We provide insight into the mechanism of dynamic instability, based on high-resolution cryo-EM structures (4.7-5.6 r A) of dynamic microtubules and microtubules stabilized by GMPCPP or Taxol. We infer that hydrolysis leads to a compaction around the E-site nucleotide at longitudinal interfaces, as well as movement of the α-tubulin intermediate domain and H7 helix. Displacement of the C-terminal helices in both α- and β-tubulin subunits suggests an effect on interactions with binding partners that contact this region. Taxol inhibits most of these conformational changes, allosterically inducing a GMPCPP-like state. Lateral interactions are similar in all conditions we examined, suggesting that microtubule lattice stability is primarily modulated at longitudinal interfaces.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Dynamic instability, the stochastic switching between growth and shrinkage, is essential for microtubule function. This behavior is driven by GTP hydrolysis in the microtubule lattice and is inhibited by anticancer agents like Taxol. We provide insight into the mechanism of dynamic instability, based on high-resolution cryo-EM structures (4.7-5.6 r A) of dynamic microtubules and microtubules stabilized by GMPCPP or Taxol. We infer that hydrolysis leads to a compaction around the E-site nucleotide at longitudinal interfaces, as well as movement of the α-tubulin intermediate domain and H7 helix. Displacement of the C-terminal helices in both α- and β-tubulin subunits suggests an effect on interactions with binding partners that contact this region. Taxol inhibits most of these conformational changes, allosterically inducing a GMPCPP-like state. Lateral interactions are similar in all conditions we examined, suggesting that microtubule lattice stability is primarily modulated at longitudinal interfaces. |
King, Chris; Garza, Esteban N; Mazor, Ronit; Linehan, Jonathan L; Pastan, Ira; Pepper, Marion; Baker, David Removing T-cell epitopes with computational protein design. Journal Article Proceedings of the National Academy of Sciences of the United States of America, 2014, ISSN: 1091-6490. @article{533, title = {Removing T-cell epitopes with computational protein design.}, author = { Chris King and Esteban N Garza and Ronit Mazor and Jonathan L Linehan and Ira Pastan and Marion Pepper and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/King_PNAS_2014A.pdf}, doi = {10.1073/pnas.1321126111}, issn = {1091-6490}, year = {2014}, date = {2014-05-01}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, abstract = {Immune responses can make protein therapeutics ineffective or even dangerous. We describe a general computational protein design method for reducing immunogenicity by eliminating known and predicted T-cell epitopes and maximizing the content of human peptide sequences without disrupting protein structure and function. We show that the method recapitulates previous experimental results on immunogenicity reduction, and we use it to disrupt T-cell epitopes in GFP and Pseudomonas exotoxin A without disrupting function.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Immune responses can make protein therapeutics ineffective or even dangerous. We describe a general computational protein design method for reducing immunogenicity by eliminating known and predicted T-cell epitopes and maximizing the content of human peptide sequences without disrupting protein structure and function. We show that the method recapitulates previous experimental results on immunogenicity reduction, and we use it to disrupt T-cell epitopes in GFP and Pseudomonas exotoxin A without disrupting function. |
Ovchinnikov, Sergey; Kamisetty, Hetunandan; Baker, David Robust and accurate prediction of residue-residue interactions across protein interfaces using evolutionary information. Journal Article eLife, 3 , pp. e02030, 2014, ISSN: 2050-084X. @article{540, title = {Robust and accurate prediction of residue-residue interactions across protein interfaces using evolutionary information.}, author = { Sergey Ovchinnikov and Hetunandan Kamisetty and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Ovchinnikov_2014A.pdf}, doi = {10.7554/eLife.02030}, issn = {2050-084X}, year = {2014}, date = {2014-05-01}, journal = {eLife}, volume = {3}, pages = {e02030}, abstract = {Do the amino acid sequence identities of residues that make contact across protein interfaces covary during evolution? If so, such covariance could be used to predict contacts across interfaces and assemble models of biological complexes. We find that residue pairs identified using a pseudo-likelihood-based method to covary across protein-protein interfaces in the 50S ribosomal unit and 28 additional bacterial protein complexes with known structure are almost always in contact in the complex, provided that the number of aligned sequences is greater than the average length of the two proteins. We use this method to make subunit contact predictions for an additional 36 protein complexes with unknown structures, and present models based on these predictions for the tripartite ATP-independent periplasmic (TRAP) transporter, the tripartite efflux system, the pyruvate formate lyase-activating enzyme complex, and the methionine ABC transporter.DOI: http://dx.doi.org/10.7554/eLife.02030.001.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Do the amino acid sequence identities of residues that make contact across protein interfaces covary during evolution? If so, such covariance could be used to predict contacts across interfaces and assemble models of biological complexes. We find that residue pairs identified using a pseudo-likelihood-based method to covary across protein-protein interfaces in the 50S ribosomal unit and 28 additional bacterial protein complexes with known structure are almost always in contact in the complex, provided that the number of aligned sequences is greater than the average length of the two proteins. We use this method to make subunit contact predictions for an additional 36 protein complexes with unknown structures, and present models based on these predictions for the tripartite ATP-independent periplasmic (TRAP) transporter, the tripartite efflux system, the pyruvate formate lyase-activating enzyme complex, and the methionine ABC transporter.DOI: http://dx.doi.org/10.7554/eLife.02030.001. |
Rajagopalan, Sridharan; Wang, Chu; Yu, Kai; Kuzin, Alexandre P; Richter, Florian; Lew, Scott; Miklos, Aleksandr E; Matthews, Megan L; Seetharaman, Jayaraman; Su, Min; Hunt, John F; Cravatt, Benjamin F; Baker, David Design of activated serine-containing catalytic triads with atomic-level accuracy Journal Article Nature chemical biology, 10 , pp. 386-391, 2014, ISSN: 1552-4469. @article{528, title = {Design of activated serine-containing catalytic triads with atomic-level accuracy}, author = { Sridharan Rajagopalan and Chu Wang and Kai Yu and Alexandre P Kuzin and Florian Richter and Scott Lew and Aleksandr E Miklos and Megan L Matthews and Jayaraman Seetharaman and Min Su and John F Hunt and Benjamin F Cravatt and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Rajagopalan_nchembio_2014A.pdf}, doi = {10.1038/nchembio.1498}, issn = {1552-4469}, year = {2014}, date = {2014-04-01}, journal = {Nature chemical biology}, volume = {10}, pages = {386-391}, abstract = {A challenge in the computational design of enzymes is that multiple properties, including substrate binding, transition state stabilization and product release, must be simultaneously optimized, and this has limited the absolute activity of successful designs. Here, we focus on a single critical property of many enzymes: the nucleophilicity of an active site residue that initiates catalysis. We design proteins with idealized serine-containing catalytic triads and assess their nucleophilicity directly in native biological systems using activity-based organophosphate probes. Crystal structures of the most successful designs show unprecedented agreement with computational models, including extensive hydrogen bonding networks between the catalytic triad (or quartet) residues, and mutagenesis experiments demonstrate that these networks are critical for serine activation and organophosphate reactivity. Following optimization by yeast display, the designs react with organophosphate probes at rates comparable to natural serine hydrolases. Co-crystal structures with diisopropyl fluorophosphate bound to the serine nucleophile suggest that the designs could provide the basis for a new class of organophosphate capture agents.}, keywords = {}, pubstate = {published}, tppubtype = {article} } A challenge in the computational design of enzymes is that multiple properties, including substrate binding, transition state stabilization and product release, must be simultaneously optimized, and this has limited the absolute activity of successful designs. Here, we focus on a single critical property of many enzymes: the nucleophilicity of an active site residue that initiates catalysis. We design proteins with idealized serine-containing catalytic triads and assess their nucleophilicity directly in native biological systems using activity-based organophosphate probes. Crystal structures of the most successful designs show unprecedented agreement with computational models, including extensive hydrogen bonding networks between the catalytic triad (or quartet) residues, and mutagenesis experiments demonstrate that these networks are critical for serine activation and organophosphate reactivity. Following optimization by yeast display, the designs react with organophosphate probes at rates comparable to natural serine hydrolases. Co-crystal structures with diisopropyl fluorophosphate bound to the serine nucleophile suggest that the designs could provide the basis for a new class of organophosphate capture agents. |
Baker, David Protein folding, structure prediction and design. Journal Article Biochemical Society transactions, 42 , pp. 225-9, 2014, ISSN: 1470-8752. @article{529, title = {Protein folding, structure prediction and design.}, author = { David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Baker_BiochemSocTrans_2014.pdf}, doi = {10.1042/BST20130055}, issn = {1470-8752}, year = {2014}, date = {2014-04-01}, journal = {Biochemical Society transactions}, volume = {42}, pages = {225-9}, abstract = {I describe how experimental studies of protein folding have led to advances in protein structure prediction and protein design. I describe the finding that protein sequences are not optimized for rapid folding, the contact order-protein folding rate correlation, the incorporation of experimental insights into protein folding into the Rosetta protein structure production methodology and the use of this methodology to determine structures from sparse experimental data. I then describe the inverse problem (protein design) and give an overview of recent work on designing proteins with new structures and functions. I also describe the contributions of the general public to these efforts through the Rosetta@home distributed computing project and the FoldIt interactive protein folding and design game.}, keywords = {}, pubstate = {published}, tppubtype = {article} } I describe how experimental studies of protein folding have led to advances in protein structure prediction and protein design. I describe the finding that protein sequences are not optimized for rapid folding, the contact order-protein folding rate correlation, the incorporation of experimental insights into protein folding into the Rosetta protein structure production methodology and the use of this methodology to determine structures from sparse experimental data. I then describe the inverse problem (protein design) and give an overview of recent work on designing proteins with new structures and functions. I also describe the contributions of the general public to these efforts through the Rosetta@home distributed computing project and the FoldIt interactive protein folding and design game. |
Wang, Yupeng; Khan, Iram F; Boissel, Sandrine; Jarjour, Jordan; Pangallo, Joseph; Thyme, Summer; Baker, David; Scharenberg, Andrew M; Rawlings, David J Progressive engineering of a homing endonuclease genome editing reagent for the murine X-linked immunodeficiency locus. Journal Article Nucleic acids research, 2014, ISSN: 1362-4962. @article{527, title = {Progressive engineering of a homing endonuclease genome editing reagent for the murine X-linked immunodeficiency locus.}, author = { Yupeng Wang and Iram F Khan and Sandrine Boissel and Jordan Jarjour and Joseph Pangallo and Summer Thyme and David Baker and Andrew M Scharenberg and David J Rawlings}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Boissel_NucleicAcids_2014.pdf}, issn = {1362-4962}, year = {2014}, date = {2014-03-01}, journal = {Nucleic acids research}, abstract = {LAGLIDADG homing endonucleases (LHEs) are compact endonucleases with 20-22 bp recognition sites, and thus are ideal scaffolds for engineering site-specific DNA cleavage enzymes for genome editing applications. Here, we describe a general approach to LHE engineering that combines rational design with directed evolution, using a yeast surface display high-throughput cleavage selection. This approach was employed to alter the binding and cleavage specificity of the I-Anil LHE to recognize a mutation in the mouse Bruton tyrosine kinase (Btk) gene causative for mouse X-linked immunodeficiency (XID)-a model of human X-linked agammaglobulinemia (XLA). The required re-targeting of I-AniI involved progressive resculpting of the DNA contact interface to accommodate nine base differences from the native cleavage sequence. The enzyme emerging from the progressive engineering process was specific for the XID mutant allele versus the wild-type (WT) allele, and exhibited activity equivalent to WT I-AniI in vitro and in cellulo reporter assays. Fusion of the enzyme to a site-specific DNA binding domain of transcription activator-like effector (TALE) resulted in a further enhancement of gene editing efficiency. These results illustrate the potential of LHE enzymes as specific and efficient tools for therapeutic genome engineering.}, keywords = {}, pubstate = {published}, tppubtype = {article} } LAGLIDADG homing endonucleases (LHEs) are compact endonucleases with 20-22 bp recognition sites, and thus are ideal scaffolds for engineering site-specific DNA cleavage enzymes for genome editing applications. Here, we describe a general approach to LHE engineering that combines rational design with directed evolution, using a yeast surface display high-throughput cleavage selection. This approach was employed to alter the binding and cleavage specificity of the I-Anil LHE to recognize a mutation in the mouse Bruton tyrosine kinase (Btk) gene causative for mouse X-linked immunodeficiency (XID)-a model of human X-linked agammaglobulinemia (XLA). The required re-targeting of I-AniI involved progressive resculpting of the DNA contact interface to accommodate nine base differences from the native cleavage sequence. The enzyme emerging from the progressive engineering process was specific for the XID mutant allele versus the wild-type (WT) allele, and exhibited activity equivalent to WT I-AniI in vitro and in cellulo reporter assays. Fusion of the enzyme to a site-specific DNA binding domain of transcription activator-like effector (TALE) resulted in a further enhancement of gene editing efficiency. These results illustrate the potential of LHE enzymes as specific and efficient tools for therapeutic genome engineering. |
Liu, Yu; Tan, Yun Lei; Zhang, Xin; Bhabha, Gira; Ekiert, Damian C; Genereux, Joseph C; Cho, Younhee; Kipnis, Yakov; Bjelic, Sinisa; Baker, David; Kelly, Jeffery W Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts. Journal Article Proceedings of the National Academy of Sciences of the United States of America, 2014, ISSN: 1091-6490. @article{526, title = {Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts.}, author = { Yu Liu and Yun Lei Tan and Xin Zhang and Gira Bhabha and Damian C Ekiert and Joseph C Genereux and Younhee Cho and Yakov Kipnis and Sinisa Bjelic and David Baker and Jeffery W Kelly}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Liu_PNAS_2014.pdf}, issn = {1091-6490}, year = {2014}, date = {2014-03-01}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, abstract = {Although much is known about protein folding in buffers, it remains unclear how the cellular protein homeostasis network functions as a system to partition client proteins between folded and functional, soluble and misfolded, and aggregated conformations. Herein, we develop small molecule folding probes that specifically react with the folded and functional fraction of the protein of interest, enabling fluorescence-based quantification of this fraction in cell lysate at a time point of interest. Importantly, these probes minimally perturb a proteintextquoterights folding equilibria within cells during and after cell lysis, because sufficient cellular chaperone/chaperonin holdase activity is created by rapid ATP depletion during cell lysis. The folding probe strategy and the faithful quantification of a particular proteintextquoterights functional fraction are exemplified with retroaldolase, a de novo designed enzyme, and transthyretin, a nonenzyme protein. Our findings challenge the often invoked assumption that the soluble fraction of a client protein is fully folded in the cell. Moreover, our results reveal that the partitioning of destabilized retroaldolase and transthyretin mutants between the aforementioned conformational states is strongly influenced by cytosolic proteostasis network perturbations. Overall, our results suggest that applying a chemical folding probe strategy to other client proteins offers opportunities to reveal how the proteostasis network functions as a system to regulate the folding and function of individual client proteins in vivo.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Although much is known about protein folding in buffers, it remains unclear how the cellular protein homeostasis network functions as a system to partition client proteins between folded and functional, soluble and misfolded, and aggregated conformations. Herein, we develop small molecule folding probes that specifically react with the folded and functional fraction of the protein of interest, enabling fluorescence-based quantification of this fraction in cell lysate at a time point of interest. Importantly, these probes minimally perturb a proteintextquoterights folding equilibria within cells during and after cell lysis, because sufficient cellular chaperone/chaperonin holdase activity is created by rapid ATP depletion during cell lysis. The folding probe strategy and the faithful quantification of a particular proteintextquoterights functional fraction are exemplified with retroaldolase, a de novo designed enzyme, and transthyretin, a nonenzyme protein. Our findings challenge the often invoked assumption that the soluble fraction of a client protein is fully folded in the cell. Moreover, our results reveal that the partitioning of destabilized retroaldolase and transthyretin mutants between the aforementioned conformational states is strongly influenced by cytosolic proteostasis network perturbations. Overall, our results suggest that applying a chemical folding probe strategy to other client proteins offers opportunities to reveal how the proteostasis network functions as a system to regulate the folding and function of individual client proteins in vivo. |
Wijma, Hein J; Floor, Robert J; Jekel, Peter A; Baker, David; Marrink, Siewert J; Janssen, Dick B Computationally designed libraries for rapid enzyme stabilization. Journal Article Protein engineering, design & selection : PEDS, 27 , pp. 49-58, 2014, ISSN: 1741-0134. @article{520, title = {Computationally designed libraries for rapid enzyme stabilization.}, author = { Hein J Wijma and Robert J Floor and Peter A Jekel and David Baker and Siewert J Marrink and Dick B Janssen}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Wijma_PEDS_2014.pdf}, doi = {10.1093/protein/gzt061}, issn = {1741-0134}, year = {2014}, date = {2014-02-01}, journal = {Protein engineering, design & selection : PEDS}, volume = {27}, pages = {49-58}, abstract = {The ability to engineer enzymes and other proteins to any desired stability would have wide-ranging applications. Here, we demonstrate that computational design of a library with chemically diverse stabilizing mutations allows the engineering of drastically stabilized and fully functional variants of the mesostable enzyme limonene epoxide hydrolase. First, point mutations were selected if they significantly improved the predicted free energy of protein folding. Disulfide bonds were designed using sampling of backbone conformational space, which tripled the number of experimentally stabilizing disulfide bridges. Next, orthogonal in silico screening steps were used to remove chemically unreasonable mutations and mutations that are predicted to increase protein flexibility. The resulting library of 64 variants was experimentally screened, which revealed 21 (pairs of) stabilizing mutations located both in relatively rigid and in flexible areas of the enzyme. Finally, combining 10-12 of these confirmed mutations resulted in multi-site mutants with an increase in apparent melting temperature from 50 to 85textdegreeC, enhanced catalytic activity, preserved regioselectivity and a >250-fold longer half-life. The developed Framework for Rapid Enzyme Stabilization by Computational libraries (FRESCO) requires far less screening than conventional directed evolution.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The ability to engineer enzymes and other proteins to any desired stability would have wide-ranging applications. Here, we demonstrate that computational design of a library with chemically diverse stabilizing mutations allows the engineering of drastically stabilized and fully functional variants of the mesostable enzyme limonene epoxide hydrolase. First, point mutations were selected if they significantly improved the predicted free energy of protein folding. Disulfide bonds were designed using sampling of backbone conformational space, which tripled the number of experimentally stabilizing disulfide bridges. Next, orthogonal in silico screening steps were used to remove chemically unreasonable mutations and mutations that are predicted to increase protein flexibility. The resulting library of 64 variants was experimentally screened, which revealed 21 (pairs of) stabilizing mutations located both in relatively rigid and in flexible areas of the enzyme. Finally, combining 10-12 of these confirmed mutations resulted in multi-site mutants with an increase in apparent melting temperature from 50 to 85textdegreeC, enhanced catalytic activity, preserved regioselectivity and a >250-fold longer half-life. The developed Framework for Rapid Enzyme Stabilization by Computational libraries (FRESCO) requires far less screening than conventional directed evolution. |
Correia, Bruno E; Bates, John T; Loomis, Rebecca J; Baneyx, Gretchen; Carrico, Chris; Jardine, Joseph G; Rupert, Peter; Correnti, Colin; Kalyuzhniy, Oleksandr; Vittal, Vinayak; Connell, Mary J; Stevens, Eric; Schroeter, Alexandria; Chen, Man; MacPherson, Skye; Serra, Andreia M; Adachi, Yumiko; Holmes, Margaret A; Li, Yuxing; Klevit, Rachel E; Graham, Barney S; Wyatt, Richard T; Baker, David; Strong, Roland K; Crowe, James E; Johnson, Philip R; Schief, William R Proof of principle for epitope-focused vaccine design Journal Article Nature, 2014, ISSN: 1476-4687. @article{522, title = {Proof of principle for epitope-focused vaccine design}, author = { Bruno E Correia and John T Bates and Rebecca J Loomis and Gretchen Baneyx and Chris Carrico and Joseph G Jardine and Peter Rupert and Colin Correnti and Oleksandr Kalyuzhniy and Vinayak Vittal and Mary J Connell and Eric Stevens and Alexandria Schroeter and Man Chen and Skye MacPherson and Andreia M Serra and Yumiko Adachi and Margaret A Holmes and Yuxing Li and Rachel E Klevit and Barney S Graham and Richard T Wyatt and David Baker and Roland K Strong and James E Crowe and Philip R Johnson and William R Schief}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Correia14.pdf}, doi = {10.1038/nature12966}, issn = {1476-4687}, year = {2014}, date = {2014-02-01}, journal = {Nature}, abstract = {Vaccines prevent infectious disease largely by inducing protective neutralizing antibodies against vulnerable epitopes. Several major pathogens have resisted traditional vaccine development, although vulnerable epitopes targeted by neutralizing antibodies have been identified for several such cases. Hence, new vaccine design methods to induce epitope-specific neutralizing antibodies are needed. Here we show, with a neutralization epitope from respiratory syncytial virus, that computational protein design can generate small, thermally and conformationally stable protein scaffolds that accurately mimic the viral epitope structure and induce potent neutralizing antibodies. These scaffolds represent promising leads for the research and development of a human respiratory syncytial virus vaccine needed to protect infants, young children and the elderly. More generally, the results provide proof of principle for epitope-focused and scaffold-based vaccine design, and encourage the evaluation and further development of these strategies for a variety of other vaccine targets, including antigenically highly variable pathogens such as human immunodeficiency virus and influenza.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Vaccines prevent infectious disease largely by inducing protective neutralizing antibodies against vulnerable epitopes. Several major pathogens have resisted traditional vaccine development, although vulnerable epitopes targeted by neutralizing antibodies have been identified for several such cases. Hence, new vaccine design methods to induce epitope-specific neutralizing antibodies are needed. Here we show, with a neutralization epitope from respiratory syncytial virus, that computational protein design can generate small, thermally and conformationally stable protein scaffolds that accurately mimic the viral epitope structure and induce potent neutralizing antibodies. These scaffolds represent promising leads for the research and development of a human respiratory syncytial virus vaccine needed to protect infants, young children and the elderly. More generally, the results provide proof of principle for epitope-focused and scaffold-based vaccine design, and encourage the evaluation and further development of these strategies for a variety of other vaccine targets, including antigenically highly variable pathogens such as human immunodeficiency virus and influenza. |
Shropshire, Tyler D; Reifert, Jack; Rajagopalan, Sridharan; Baker, David; Feinstein, Stuart C; Daugherty, Patrick S Amyloid β peptide cleavage by kallikrein 7 attenuates fibril growth and rescues neurons from Aβ-mediated toxicity in vitro. Journal Article Biological chemistry, 395 , pp. 109-18, 2014, ISSN: 1437-4315. @article{510, title = {Amyloid β peptide cleavage by kallikrein 7 attenuates fibril growth and rescues neurons from Aβ-mediated toxicity in vitro.}, author = { Tyler D Shropshire and Jack Reifert and Sridharan Rajagopalan and David Baker and Stuart C Feinstein and Patrick S Daugherty}, doi = {10.1515/hsz-2013-0230}, issn = {1437-4315}, year = {2014}, date = {2014-01-01}, journal = {Biological chemistry}, volume = {395}, pages = {109-18}, abstract = {Abstract The gradual accumulation and assembly of β-amyloid (Aβ) peptide into neuritic plaques is a major pathological hallmark of Alzheimer disease (AD). Proteolytic degradation of Aβ is an important clearance mechanism under normal circumstances, and it has been found to be compromised in those with AD. Here, the extended substrate specificity and Aβ-degrading capacity of kallikrein 7 (KLK7), a serine protease with a unique chymotrypsin-like specificity, was characterized. Preferred peptide substrates of KLK7 identified using a bacterial display substrate library were found to exhibit a consensus motif of RXΦ(Y/F)textdownarrow(Y/F)textdownarrow(S/A/G/T) or RXΦ(Y/F)textdownarrow(S/T/A) (Φ=hydrophobic), which is remarkably similar to the hydrophobic core motif of Aβ (K16L17V18F19F20 A21) that is largely responsible for aggregation propensity. KLK7 was found to cleave after both Phe residues within the core of Aβ42 in vitro, thereby inhibiting Aβ fibril formation and promoting the degradation of preformed fibrils. Finally, the treatment of Aβ oligomer preparations with KLK7, but not inactive pro-KLK7, significantly reduced Aβ42-mediated toxicity to rat hippocampal neurons to the same extent as the known Aβ-degrading protease insulin-degrading enzyme (IDE). Taken together, these results indicate that KLK7 possesses an Aβ-degrading capacity that can ameliorate the toxic effects of the aggregated peptide in vitro.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Abstract The gradual accumulation and assembly of β-amyloid (Aβ) peptide into neuritic plaques is a major pathological hallmark of Alzheimer disease (AD). Proteolytic degradation of Aβ is an important clearance mechanism under normal circumstances, and it has been found to be compromised in those with AD. Here, the extended substrate specificity and Aβ-degrading capacity of kallikrein 7 (KLK7), a serine protease with a unique chymotrypsin-like specificity, was characterized. Preferred peptide substrates of KLK7 identified using a bacterial display substrate library were found to exhibit a consensus motif of RXΦ(Y/F)textdownarrow(Y/F)textdownarrow(S/A/G/T) or RXΦ(Y/F)textdownarrow(S/T/A) (Φ=hydrophobic), which is remarkably similar to the hydrophobic core motif of Aβ (K16L17V18F19F20 A21) that is largely responsible for aggregation propensity. KLK7 was found to cleave after both Phe residues within the core of Aβ42 in vitro, thereby inhibiting Aβ fibril formation and promoting the degradation of preformed fibrils. Finally, the treatment of Aβ oligomer preparations with KLK7, but not inactive pro-KLK7, significantly reduced Aβ42-mediated toxicity to rat hippocampal neurons to the same extent as the known Aβ-degrading protease insulin-degrading enzyme (IDE). Taken together, these results indicate that KLK7 possesses an Aβ-degrading capacity that can ameliorate the toxic effects of the aggregated peptide in vitro. |
Thyme, Summer; Baker, David Redesigning the Specificity of Protein-DNA Interactions with Rosetta. Journal Article Methods in molecular biology (Clifton, N.J.), 1123 , pp. 265-82, 2014, ISSN: 1940-6029. @article{525, title = {Redesigning the Specificity of Protein-DNA Interactions with Rosetta.}, author = { Summer Thyme and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Thyme_2014.pdf}, doi = {10.1007/978-1-62703-968-0_17}, issn = {1940-6029}, year = {2014}, date = {2014-01-01}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {1123}, pages = {265-82}, abstract = {Building protein tools that can selectively bind or cleave specific DNA sequences requires efficient technologies for modifying protein-DNA interactions. Computational design is one method for accomplishing this goal. In this chapter, we present the current state of protein-DNA interface design with the Rosetta macromolecular modeling program. The LAGLIDADG endonuclease family of DNA-cleaving enzymes, under study as potential gene therapy reagents, has been the main testing ground for these in silico protocols. At this time, the computational methods are most useful for designing endonuclease variants that can accommodate small numbers of target site substitutions. Attempts to engineer for more extensive interface changes will likely benefit from an approach that uses the computational design results in conjunction with a high-throughput directed evolution or screening procedure. The family of enzymes presents an engineering challenge because their interfaces are highly integrated and there is significant coordination between the binding and catalysis events. Future developments in the computational algorithms depend on experimental feedback to improve understanding and modeling of these complex enzymatic features. This chapter presents both the basic method of design that has been successfully used to modulate specificity and more advanced procedures that incorporate DNA flexibility and other properties that are likely necessary for reliable modeling of more extensive target site changes.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Building protein tools that can selectively bind or cleave specific DNA sequences requires efficient technologies for modifying protein-DNA interactions. Computational design is one method for accomplishing this goal. In this chapter, we present the current state of protein-DNA interface design with the Rosetta macromolecular modeling program. The LAGLIDADG endonuclease family of DNA-cleaving enzymes, under study as potential gene therapy reagents, has been the main testing ground for these in silico protocols. At this time, the computational methods are most useful for designing endonuclease variants that can accommodate small numbers of target site substitutions. Attempts to engineer for more extensive interface changes will likely benefit from an approach that uses the computational design results in conjunction with a high-throughput directed evolution or screening procedure. The family of enzymes presents an engineering challenge because their interfaces are highly integrated and there is significant coordination between the binding and catalysis events. Future developments in the computational algorithms depend on experimental feedback to improve understanding and modeling of these complex enzymatic features. This chapter presents both the basic method of design that has been successfully used to modulate specificity and more advanced procedures that incorporate DNA flexibility and other properties that are likely necessary for reliable modeling of more extensive target site changes. |
Mao, Binchen; Tejero, Roberto; Baker, David; Montelione, Gaetano Thomas Protein NMR Structures Refined with Rosetta Have Higher Accuracy Relative to Corresponding X-ray Crystal Structures. Journal Article Journal of the American Chemical Society, 2014, ISSN: 1520-5126. @article{519, title = {Protein NMR Structures Refined with Rosetta Have Higher Accuracy Relative to Corresponding X-ray Crystal Structures.}, author = { Binchen Mao and Roberto Tejero and David Baker and Gaetano Thomas Montelione}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Mao_JACS_2014.pdf}, issn = {1520-5126}, year = {2014}, date = {2014-01-01}, journal = {Journal of the American Chemical Society}, abstract = {We have found that refinement of protein NMR structures using Rosetta with experimental NMR restraints yields more accurate protein NMR structures than those that have been deposited in the PDB using standard refinement protocols. Using 40 pairs of NMR and X-ray crystal structures determined by the Northeast Structural Genomics Consortium, for proteins ranging in size from 5 - 22 kDa, restrained-Rosetta refined structures fit better to the raw experimental data, are in better agreement with their X-ray counterparts, and have better phasing power compared to conventionally determined NMR structures. For 38 proteins for which NMR ensembles were available and which had similar structures in solution and in the crystal, all of the restrained-Rosetta refined NMR structures were sufficiently accurate to be used for solving the corresponding X-ray crystal structures by molecular replacement. The protocol for restrained refinement of protein NMR structures was also compared with restrained CS-Rosetta calculations. For proteins smaller than 10 kDa, restrained CS-Rosetta, starting from extended conformations, provides slightly more accurate structures, while for proteins in the size range of 10 - 25 kDa the less cpu intensive restrained-Rosetta refinement protocols provided more accurate structures. The restrained-Rosetta protocols described here can improve the accuracy of protein NMR structures, and should find broad and general for studies of protein structure and function.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We have found that refinement of protein NMR structures using Rosetta with experimental NMR restraints yields more accurate protein NMR structures than those that have been deposited in the PDB using standard refinement protocols. Using 40 pairs of NMR and X-ray crystal structures determined by the Northeast Structural Genomics Consortium, for proteins ranging in size from 5 - 22 kDa, restrained-Rosetta refined structures fit better to the raw experimental data, are in better agreement with their X-ray counterparts, and have better phasing power compared to conventionally determined NMR structures. For 38 proteins for which NMR ensembles were available and which had similar structures in solution and in the crystal, all of the restrained-Rosetta refined NMR structures were sufficiently accurate to be used for solving the corresponding X-ray crystal structures by molecular replacement. The protocol for restrained refinement of protein NMR structures was also compared with restrained CS-Rosetta calculations. For proteins smaller than 10 kDa, restrained CS-Rosetta, starting from extended conformations, provides slightly more accurate structures, while for proteins in the size range of 10 - 25 kDa the less cpu intensive restrained-Rosetta refinement protocols provided more accurate structures. The restrained-Rosetta protocols described here can improve the accuracy of protein NMR structures, and should find broad and general for studies of protein structure and function. |
Demers, Jean-Philippe; Habenstein, Birgit; Loquet, Antoine; Vasa, Suresh Kumar; Giller, Karin; Becker, Stefan; Baker, David; Lange, Adam; Sgourakis, Nikolaos G High-resolution structure of the Shigella type-III secretion needle by solid-state NMR and cryo-electron microscopy. Journal Article Nature communications, 5 , pp. 4976, 2014, ISSN: 2041-1723. @article{620, title = {High-resolution structure of the Shigella type-III secretion needle by solid-state NMR and cryo-electron microscopy.}, author = { Jean-Philippe Demers and Birgit Habenstein and Antoine Loquet and Suresh Kumar Vasa and Karin Giller and Stefan Becker and David Baker and Adam Lange and Nikolaos G Sgourakis}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Demers_Nature_2014.pdf}, doi = {10.1038/ncomms5976}, issn = {2041-1723}, year = {2014}, date = {2014-00-01}, journal = {Nature communications}, volume = {5}, pages = {4976}, abstract = {We introduce a general hybrid approach for determining the structures of supramolecular assemblies. Cryo-electron microscopy (cryo-EM) data define the overall envelope of the assembly and rigid-body orientation of the subunits while solid-state nuclear magnetic resonance (ssNMR) chemical shifts and distance constraints define the local secondary structure, protein fold and inter-subunit interactions. Finally, Rosetta structure calculations provide a general framework to integrate the different sources of structural information. Combining a 7.7-r A cryo-EM density map and 996 ssNMR distance constraints, the structure of the type-III secretion system needle of Shigella flexneri is determined to a precision of 0.4 r A. The calculated structures are cross-validated using an independent data set of 691 ssNMR constraints and scanning transmission electron microscopy measurements. The hybrid model resolves the conformation of the non-conserved N terminus, which occupies a protrusion in the cryo-EM density, and reveals conserved pore residues forming a continuous pattern of electrostatic interactions, thereby suggesting a mechanism for effector protein translocation.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We introduce a general hybrid approach for determining the structures of supramolecular assemblies. Cryo-electron microscopy (cryo-EM) data define the overall envelope of the assembly and rigid-body orientation of the subunits while solid-state nuclear magnetic resonance (ssNMR) chemical shifts and distance constraints define the local secondary structure, protein fold and inter-subunit interactions. Finally, Rosetta structure calculations provide a general framework to integrate the different sources of structural information. Combining a 7.7-r A cryo-EM density map and 996 ssNMR distance constraints, the structure of the type-III secretion system needle of Shigella flexneri is determined to a precision of 0.4 r A. The calculated structures are cross-validated using an independent data set of 691 ssNMR constraints and scanning transmission electron microscopy measurements. The hybrid model resolves the conformation of the non-conserved N terminus, which occupies a protrusion in the cryo-EM density, and reveals conserved pore residues forming a continuous pattern of electrostatic interactions, thereby suggesting a mechanism for effector protein translocation. |
Leblanc, Pierre; Moise, Leonard; Luza, Cybelle; Chantaralawan, Kanawat; Lezeau, Lynchy; Yuan, Jianping; Field, Mary; Richer, Daniel; Boyle, Christine; Martin, William D; Fishman, Jordan B; Berg, Eric A; Baker, David; Zeigler, Brandon; Mais, Dale E; Taylor, William; Coleman, Russell; Warren, Shaw H; Gelfand, Jeffrey A; Groot, Anne De S; Brauns, Timothy; Poznansky, Mark C VaxCelerate II: rapid development of a self-assembling vaccine for Lassa fever. Journal Article Human vaccines & immunotherapeutics, 10 , pp. 3022-38, 2014, ISSN: 2164-554X. @article{618, title = {VaxCelerate II: rapid development of a self-assembling vaccine for Lassa fever.}, author = { Pierre Leblanc and Leonard Moise and Cybelle Luza and Kanawat Chantaralawan and Lynchy Lezeau and Jianping Yuan and Mary Field and Daniel Richer and Christine Boyle and William D Martin and Jordan B Fishman and Eric A Berg and David Baker and Brandon Zeigler and Dale E Mais and William Taylor and Russell Coleman and H Shaw Warren and Jeffrey A Gelfand and Anne S De Groot and Timothy Brauns and Mark C Poznansky}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Leblanc_HumanVI_2014.pdf}, doi = {10.4161/hv.34413}, issn = {2164-554X}, year = {2014}, date = {2014-00-01}, journal = {Human vaccines & immunotherapeutics}, volume = {10}, pages = {3022-38}, abstract = {Development of effective vaccines against emerging infectious diseases (EID) can take as much or more than a decade to progress from pathogen isolation/identification to clinical approval. As a result, conventional approaches fail to produce field-ready vaccines before the EID has spread extensively. Lassa is a prototypical emerging infectious disease endemic to West Africa for which no successful vaccine is available. We established the VaxCelerate Consortium to address the need for more rapid vaccine development by creating a platform capable of generating and pre-clinically testing a new vaccine against specific pathogen targets in less than 120 d A self-assembling vaccine is at the core of the approach. It consists of a fusion protein composed of the immunostimulatory Mycobacterium tuberculosis heat shock protein 70 (MtbHSP70) and the biotin binding protein, avidin. Mixing the resulting protein (MAV) with biotinylated pathogen-specific immunogenic peptides yields a self-assembled vaccine (SAV). To meet the time constraint imposed on this project, we used a distributed R&D model involving experts in the fields of protein engineering and production, bioinformatics, peptide synthesis/design and GMP/GLP manufacturing and testing standards. SAV immunogenicity was first tested using H1N1 influenza specific peptides and the entire VaxCelerate process was then tested in a mock live-fire exercise targeting Lassa fever virus. We demonstrated that the Lassa fever vaccine induced significantly increased class II peptide specific interferon-γ CD4(+) T cell responses in HLA-DR3 transgenic mice compared to peptide or MAV alone controls. We thereby demonstrated that our SAV in combination with a distributed development model may facilitate accelerated regulatory review by using an identical design for each vaccine and by applying safety and efficacy assessment tools that are more relevant to human vaccine responses than current animal models.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Development of effective vaccines against emerging infectious diseases (EID) can take as much or more than a decade to progress from pathogen isolation/identification to clinical approval. As a result, conventional approaches fail to produce field-ready vaccines before the EID has spread extensively. Lassa is a prototypical emerging infectious disease endemic to West Africa for which no successful vaccine is available. We established the VaxCelerate Consortium to address the need for more rapid vaccine development by creating a platform capable of generating and pre-clinically testing a new vaccine against specific pathogen targets in less than 120 d A self-assembling vaccine is at the core of the approach. It consists of a fusion protein composed of the immunostimulatory Mycobacterium tuberculosis heat shock protein 70 (MtbHSP70) and the biotin binding protein, avidin. Mixing the resulting protein (MAV) with biotinylated pathogen-specific immunogenic peptides yields a self-assembled vaccine (SAV). To meet the time constraint imposed on this project, we used a distributed R&D model involving experts in the fields of protein engineering and production, bioinformatics, peptide synthesis/design and GMP/GLP manufacturing and testing standards. SAV immunogenicity was first tested using H1N1 influenza specific peptides and the entire VaxCelerate process was then tested in a mock live-fire exercise targeting Lassa fever virus. We demonstrated that the Lassa fever vaccine induced significantly increased class II peptide specific interferon-γ CD4(+) T cell responses in HLA-DR3 transgenic mice compared to peptide or MAV alone controls. We thereby demonstrated that our SAV in combination with a distributed development model may facilitate accelerated regulatory review by using an identical design for each vaccine and by applying safety and efficacy assessment tools that are more relevant to human vaccine responses than current animal models. |
2013 |
Strauch, Eva-Maria; Fleishman, Sarel J; Baker, David Computational design of a pH-sensitive IgG binding protein Journal Article Proceedings of the National Academy of Sciences of the United States of America, 2013, ISSN: 1091-6490. @article{499, title = {Computational design of a pH-sensitive IgG binding protein}, author = { Eva-Maria Strauch and Sarel J Fleishman and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Strauch-1313605111_PNAS_13W.pdf}, issn = {1091-6490}, year = {2013}, date = {2013-12-01}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, abstract = {Computational design provides the opportunity to program protein-protein interactions for desired applications. We used de novo protein interface design to generate a pH-dependent Fc domain binding protein that buries immunoglobulin G (IgG) His-433. Using next-generation sequencing of na"ive and selected pools of a library of design variants, we generated a molecular footprint of the designed binding surface, confirming the binding mode and guiding further optimization of the balance between affinity and pH sensitivity. In biolayer interferometry experiments, the optimized design binds IgG with a Kd of ~4 nM at pH 8.2, and approximately 500-fold more weakly at pH 5.5. The protein is extremely stable, heat-resistant and highly expressed in bacteria, and allows pH-based control of binding for IgG affinity purification and diagnostic devices.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Computational design provides the opportunity to program protein-protein interactions for desired applications. We used de novo protein interface design to generate a pH-dependent Fc domain binding protein that buries immunoglobulin G (IgG) His-433. Using next-generation sequencing of na"ive and selected pools of a library of design variants, we generated a molecular footprint of the designed binding surface, confirming the binding mode and guiding further optimization of the balance between affinity and pH sensitivity. In biolayer interferometry experiments, the optimized design binds IgG with a Kd of ~4 nM at pH 8.2, and approximately 500-fold more weakly at pH 5.5. The protein is extremely stable, heat-resistant and highly expressed in bacteria, and allows pH-based control of binding for IgG affinity purification and diagnostic devices. |
Fang, Jie; Mehlich, Alexander; Koga, Nobuyasu; Huang, Jiqing; Koga, Rie; Gao, Xiaoye; Hu, Chunguang; Jin, Chi; Rief, Matthias; Kast, Juergen; Baker, David; Li, Hongbin Forced protein unfolding leads to highly elastic and tough protein hydrogels. Journal Article Nature communications, 4 , pp. 2974, 2013, ISSN: 2041-1723. @article{517, title = {Forced protein unfolding leads to highly elastic and tough protein hydrogels.}, author = { Jie Fang and Alexander Mehlich and Nobuyasu Koga and Jiqing Huang and Rie Koga and Xiaoye Gao and Chunguang Hu and Chi Jin and Matthias Rief and Juergen Kast and David Baker and Hongbin Li}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Fang_NatCommun_2013.pdf}, doi = {10.1038/ncomms3974}, issn = {2041-1723}, year = {2013}, date = {2013-12-01}, journal = {Nature communications}, volume = {4}, pages = {2974}, abstract = {Protein-based hydrogels usually do not exhibit high stretchability or toughness, significantly limiting the scope of their potential biomedical applications. Here we report the engineering of a chemically cross-linked, highly elastic and tough protein hydrogel using a mechanically extremely labile, de novo-designed protein that assumes the classical ferredoxin-like fold structure. Due to the low mechanical stability of the ferredoxin-like fold structure, swelling of hydrogels causes a significant fraction of the folded domains to unfold. Subsequent collapse and aggregation of unfolded ferredoxin-like domains leads to intertwining of physically and chemically cross-linked networks, entailing hydrogels with unusual physical and mechanical properties: a negative swelling ratio, high stretchability and toughness. These hydrogels can withstand an average strain of 450% before breaking and show massive energy dissipation. Upon relaxation, refolding of the ferredoxin-like domains enables the hydrogel to recover its massive hysteresis. This novel biomaterial may expand the scope of hydrogel applications in tissue engineering.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Protein-based hydrogels usually do not exhibit high stretchability or toughness, significantly limiting the scope of their potential biomedical applications. Here we report the engineering of a chemically cross-linked, highly elastic and tough protein hydrogel using a mechanically extremely labile, de novo-designed protein that assumes the classical ferredoxin-like fold structure. Due to the low mechanical stability of the ferredoxin-like fold structure, swelling of hydrogels causes a significant fraction of the folded domains to unfold. Subsequent collapse and aggregation of unfolded ferredoxin-like domains leads to intertwining of physically and chemically cross-linked networks, entailing hydrogels with unusual physical and mechanical properties: a negative swelling ratio, high stretchability and toughness. These hydrogels can withstand an average strain of 450% before breaking and show massive energy dissipation. Upon relaxation, refolding of the ferredoxin-like domains enables the hydrogel to recover its massive hysteresis. This novel biomaterial may expand the scope of hydrogel applications in tissue engineering. |
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 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. |
Thyme, Summer B; Boissel, Sandrine J S; Quadri, Arshiya S; Nolan, Tony; Baker, Dean A; Park, Rachel U; Kusak, Lara; Ashworth, Justin; Baker, David Reprogramming homing endonuclease specificity through computational design and directed evolution. Journal Article Nucleic acids research, 2013, ISSN: 1362-4962. @article{515, title = {Reprogramming homing endonuclease specificity through computational design and directed evolution.}, author = { Summer B Thyme and Sandrine J S Boissel and S Arshiya Quadri and Tony Nolan and Dean A Baker and Rachel U Park and Lara Kusak and Justin Ashworth and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Thyme_NAR_2013.pdf}, issn = {1362-4962}, year = {2013}, date = {2013-11-01}, journal = {Nucleic acids research}, abstract = {Homing endonucleases (HEs) can be used to induce targeted genome modification to reduce the fitness of pathogen vectors such as the malaria-transmitting Anopheles gambiae and to correct deleterious mutations in genetic diseases. We describe the creation of an extensive set of HE variants with novel DNA cleavage specificities using an integrated experimental and computational approach. Using computational modeling and an improved selection strategy, which optimizes specificity in addition to activity, we engineered an endonuclease to cleave in a gene associated with Anopheles sterility and another to cleave near a mutation that causes pyruvate kinase deficiency. In the course of this work we observed unanticipated context-dependence between bases which will need to be mechanistically understood for reprogramming of specificity to succeed more generally.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Homing endonucleases (HEs) can be used to induce targeted genome modification to reduce the fitness of pathogen vectors such as the malaria-transmitting Anopheles gambiae and to correct deleterious mutations in genetic diseases. We describe the creation of an extensive set of HE variants with novel DNA cleavage specificities using an integrated experimental and computational approach. Using computational modeling and an improved selection strategy, which optimizes specificity in addition to activity, we engineered an endonuclease to cleave in a gene associated with Anopheles sterility and another to cleave near a mutation that causes pyruvate kinase deficiency. In the course of this work we observed unanticipated context-dependence between bases which will need to be mechanistically understood for reprogramming of specificity to succeed more generally. |
Cherny, Izhack; Greisen, Per; Ashani, Yacov; Khare, Sagar D; Oberdorfer, Gustav; Leader, Haim; Baker, David; Tawfik, Dan S Engineering V-type nerve agents detoxifying enzymes using computationally focused libraries Journal Article ACS chemical biology, 8 , pp. 2394-403, 2013, ISSN: 1554-8937. @article{497, title = {Engineering V-type nerve agents detoxifying enzymes using computationally focused libraries}, author = { Izhack Cherny and Per Greisen and Yacov Ashani and Sagar D Khare and Gustav Oberdorfer and Haim Leader and David Baker and Dan S Tawfik}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Cherny_cb4004892_13W.pdf}, doi = {10.1021/cb4004892}, issn = {1554-8937}, year = {2013}, date = {2013-11-01}, journal = {ACS chemical biology}, volume = {8}, pages = {2394-403}, abstract = {VX and its Russian (RVX) and Chinese (CVX) analogues rapidly inactivate acetylcholinesterase and are the most toxic stockpile nerve agents. These organophosphates have a thiol leaving group with a choline-like moiety and are hydrolyzed very slowly by natural enzymes. We used an integrated computational and experimental approach to increase Brevundimonas diminuta phosphotriesterasetextquoterights (PTE) detoxification rate of V-agents by 5000-fold. Computational models were built of the complex between PTE and V-agents. On the basis of these models, the active site was redesigned to be complementary in shape to VX and RVX and to include favorable electrostatic interactions with their choline-like leaving group. Small libraries based on designed sequences were constructed. The libraries were screened by a direct assay for V-agent detoxification, as our initial studies showed that colorimetric surrogates fail to report the detoxification rates of the actual agents. The experimental results were fed back to improve the computational models. Overall, five rounds of iterating between experiment and model refinement led to variants that hydrolyze the toxic SP isomers of all three V-agents with kcat/KM values of up to 5 texttimes 10(6) M(-1) min(-1) and also efficiently detoxify G-agents. These new catalysts provide the basis for broad spectrum nerve agent detoxification.}, keywords = {}, pubstate = {published}, tppubtype = {article} } VX and its Russian (RVX) and Chinese (CVX) analogues rapidly inactivate acetylcholinesterase and are the most toxic stockpile nerve agents. These organophosphates have a thiol leaving group with a choline-like moiety and are hydrolyzed very slowly by natural enzymes. We used an integrated computational and experimental approach to increase Brevundimonas diminuta phosphotriesterasetextquoterights (PTE) detoxification rate of V-agents by 5000-fold. Computational models were built of the complex between PTE and V-agents. On the basis of these models, the active site was redesigned to be complementary in shape to VX and RVX and to include favorable electrostatic interactions with their choline-like leaving group. Small libraries based on designed sequences were constructed. The libraries were screened by a direct assay for V-agent detoxification, as our initial studies showed that colorimetric surrogates fail to report the detoxification rates of the actual agents. The experimental results were fed back to improve the computational models. Overall, five rounds of iterating between experiment and model refinement led to variants that hydrolyze the toxic SP isomers of all three V-agents with kcat/KM values of up to 5 texttimes 10(6) M(-1) min(-1) and also efficiently detoxify G-agents. These new catalysts provide the basis for broad spectrum nerve agent detoxification. |
Moretti, Rocco; Fleishman, Sarel J; Agius, Rudi; Torchala, Mieczyslaw; Bates, Paul A; Kastritis, Panagiotis L; Rodrigues, Jo~ao P G L M; Trellet, Mika"el; Bonvin, Alexandre M J J; Cui, Meng; Rooman, Marianne; Gillis, Dimitri; Dehouck, Yves; Moal, Iain; Romero-Durana, Miguel; Perez-Cano, Laura; Pallara, Chiara; Jimenez, Brian; Fernandez-Recio, Juan; Flores, Samuel; Pacella, Michael; Kilambi, Krishna Praneeth; Gray, Jeffrey J; Popov, Petr; Grudinin, Sergei; Esquivel-Rodr'iguez, Juan; Kihara, Daisuke; Zhao, Nan; Korkin, Dmitry; Zhu, Xiaolei; Demerdash, Omar N A; Mitchell, Julie C; Kanamori, Eiji; Tsuchiya, Yuko; Nakamura, Haruki; Lee, Hasup; Park, Hahnbeom; Seok, Chaok; Sarmiento, Jamica; Liang, Shide; Teraguchi, Shusuke; Standley, Daron M; Shimoyama, Hiromitsu; Terashi, Genki; Takeda-Shitaka, Mayuko; Iwadate, Mitsuo; Umeyama, Hideaki; Beglov, Dmitri; Hall, David R; Kozakov, Dima; Vajda, Sandor; Pierce, Brian G; Hwang, Howook; Vreven, Thom; Weng, Zhiping; Huang, Yangyu; Li, Haotian; Yang, Xiufeng; Ji, Xiaofeng; Liu, Shiyong; Xiao, Yi; Zacharias, Martin; Qin, Sanbo; Zhou, Huan-Xiang; Huang, Sheng-You; Zou, Xiaoqin; Velankar, Sameer; Janin, Jo"el; Wodak, Shoshana J; Baker, David Community-wide evaluation of methods for predicting the effect of mutations on protein-protein interactions. Journal Article Proteins, 81 , pp. 1980-7, 2013, ISSN: 1097-0134. @article{505, title = {Community-wide evaluation of methods for predicting the effect of mutations on protein-protein interactions.}, author = { Rocco Moretti and Sarel J Fleishman and Rudi Agius and Mieczyslaw Torchala and Paul A Bates and Panagiotis L Kastritis and Jo~ao P G L M Rodrigues and Mika"el Trellet and Alexandre M J J Bonvin and Meng Cui and Marianne Rooman and Dimitri Gillis and Yves Dehouck and Iain Moal and Miguel Romero-Durana and Laura Perez-Cano and Chiara Pallara and Brian Jimenez and Juan Fernandez-Recio and Samuel Flores and Michael Pacella and Krishna Praneeth Kilambi and Jeffrey J Gray and Petr Popov and Sergei Grudinin and Juan Esquivel-Rodr'iguez and Daisuke Kihara and Nan Zhao and Dmitry Korkin and Xiaolei Zhu and Omar N A Demerdash and Julie C Mitchell and Eiji Kanamori and Yuko Tsuchiya and Haruki Nakamura and Hasup Lee and Hahnbeom Park and Chaok Seok and Jamica Sarmiento and Shide Liang and Shusuke Teraguchi and Daron M Standley and Hiromitsu Shimoyama and Genki Terashi and Mayuko Takeda-Shitaka and Mitsuo Iwadate and Hideaki Umeyama and Dmitri Beglov and David R Hall and Dima Kozakov and Sandor Vajda and Brian G Pierce and Howook Hwang and Thom Vreven and Zhiping Weng and Yangyu Huang and Haotian Li and Xiufeng Yang and Xiaofeng Ji and Shiyong Liu and Yi Xiao and Martin Zacharias and Sanbo Qin and Huan-Xiang Zhou and Sheng-You Huang and Xiaoqin Zou and Sameer Velankar and Jo"el Janin and Shoshana J Wodak and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Moretti_Proteins_2013.pdf}, doi = {10.1002/prot.24356}, issn = {1097-0134}, year = {2013}, date = {2013-11-01}, journal = {Proteins}, volume = {81}, pages = {1980-7}, abstract = {Community-wide blind prediction experiments such as CAPRI and CASP provide an objective measure of the current state of predictive methodology. Here we describe a community-wide assessment of methods to predict the effects of mutations on protein-protein interactions. Twenty-two groups predicted the effects of comprehensive saturation mutagenesis for two designed influenza hemagglutinin binders and the results were compared with experimental yeast display enrichment data obtained using deep sequencing. The most successful methods explicitly considered the effects of mutation on monomer stability in addition to binding affinity, carried out explicit side-chain sampling and backbone relaxation, evaluated packing, electrostatic, and solvation effects, and correctly identified around a third of the beneficial mutations. Much room for improvement remains for even the best techniques, and large-scale fitness landscapes should continue to provide an excellent test bed for continued evaluation of both existing and new prediction methodologies.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Community-wide blind prediction experiments such as CAPRI and CASP provide an objective measure of the current state of predictive methodology. Here we describe a community-wide assessment of methods to predict the effects of mutations on protein-protein interactions. Twenty-two groups predicted the effects of comprehensive saturation mutagenesis for two designed influenza hemagglutinin binders and the results were compared with experimental yeast display enrichment data obtained using deep sequencing. The most successful methods explicitly considered the effects of mutation on monomer stability in addition to binding affinity, carried out explicit side-chain sampling and backbone relaxation, evaluated packing, electrostatic, and solvation effects, and correctly identified around a third of the beneficial mutations. Much room for improvement remains for even the best techniques, and large-scale fitness landscapes should continue to provide an excellent test bed for continued evaluation of both existing and new prediction methodologies. |
DiMaio, Frank; Echols, Nathaniel; Headd, Jeffrey J; Terwilliger, Thomas C; Adams, Paul D; Baker, David Improved low-resolution crystallographic refinement with Phenix and Rosetta. Journal Article Nature methods, 10 , pp. 1102-4, 2013, ISSN: 1548-7105. @article{513, title = {Improved low-resolution crystallographic refinement with Phenix and Rosetta.}, author = { Frank DiMaio and Nathaniel Echols and Jeffrey J Headd and Thomas C Terwilliger and Paul D Adams and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/DiMaio_NatureMethods_2013.pdf}, doi = {10.1038/nmeth.2648}, issn = {1548-7105}, year = {2013}, date = {2013-11-01}, journal = {Nature methods}, volume = {10}, pages = {1102-4}, abstract = {Refinement of macromolecular structures against low-resolution crystallographic data is limited by the ability of current methods to converge on a structure with realistic geometry. We developed a low-resolution crystallographic refinement method that combines the Rosetta sampling methodology and energy function with reciprocal-space X-ray refinement in Phenix. On a set of difficult low-resolution cases, the method yielded improved model geometry and lower free R factors than alternate refinement methods.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Refinement of macromolecular structures against low-resolution crystallographic data is limited by the ability of current methods to converge on a structure with realistic geometry. We developed a low-resolution crystallographic refinement method that combines the Rosetta sampling methodology and energy function with reciprocal-space X-ray refinement in Phenix. On a set of difficult low-resolution cases, the method yielded improved model geometry and lower free R factors than alternate refinement methods. |
Song, Yifan; DiMaio, Frank; Wang, Ray Yu-Ruei; Kim, David; Miles, Chris; Brunette, Tj; Thompson, James; Baker, David High-resolution comparative modeling with RosettaCM. Journal Article Structure (London, England : 1993), 21 , pp. 1735-42, 2013, ISSN: 1878-4186. @article{512, title = {High-resolution comparative modeling with RosettaCM.}, author = { Yifan Song and Frank DiMaio and Ray Yu-Ruei Wang and David Kim and Chris Miles and Tj Brunette and James Thompson and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Song_Structure_2013.pdf}, doi = {10.1016/j.str.2013.08.005}, issn = {1878-4186}, year = {2013}, date = {2013-10-01}, journal = {Structure (London, England : 1993)}, volume = {21}, pages = {1735-42}, abstract = {We describe an improved method for comparative modeling, RosettaCM, which optimizes a physically realistic all-atom energy function over the conformational space defined by homologous structures. Given a set of sequence alignments, RosettaCM assembles topologies by recombining aligned segments in Cartesian space and building unaligned regions de novo in torsion space. The junctions between segments are regularized using a loop closure method combining fragment superposition with gradient-based minimization. The energies of the resulting models are optimized by all-atom refinement, and the most representative low-energy model is selected. The CASP10 experiment suggests that RosettaCM yields models with more accurate side-chain and backbone conformations than other methods when the sequence identity to the templates is greater than ~15%.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We describe an improved method for comparative modeling, RosettaCM, which optimizes a physically realistic all-atom energy function over the conformational space defined by homologous structures. Given a set of sequence alignments, RosettaCM assembles topologies by recombining aligned segments in Cartesian space and building unaligned regions de novo in torsion space. The junctions between segments are regularized using a loop closure method combining fragment superposition with gradient-based minimization. The energies of the resulting models are optimized by all-atom refinement, and the most representative low-energy model is selected. The CASP10 experiment suggests that RosettaCM yields models with more accurate side-chain and backbone conformations than other methods when the sequence identity to the templates is greater than ~15%. |
Niv'on, Lucas G; Bjelic, Sinisa; King, Chris; Baker, David Automating human intuition for protein design Journal Article Proteins, 2013, ISSN: 1097-0134. @article{495, title = {Automating human intuition for protein design}, author = { Lucas G Niv'on and Sinisa Bjelic and Chris King and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2015/12/Nivón_prot24463_13M.pdf}, doi = {10.1002/prot.24463}, issn = {1097-0134}, year = {2013}, date = {2013-10-01}, journal = {Proteins}, abstract = {In the design of new enzymes and binding proteins, human intuition is often used to modify computationally designed amino acid sequences prior to experimental characterization. The manual sequence changes involve both reversions of amino acid mutations back to the identity present in the parent scaffold and the introduction of residues making additional interactions with the binding partner or backing up first shell interactions. Automation of this manual sequence refinement process would allow more systematic evaluation and considerably reduce the amount of human designer effort involved. Here we introduce a benchmark for evaluating the ability of automated methods to recapitulate the sequence changes made to computer-generated models by human designers, and use it to assess alternative computational methods. We find the best performance for a greedy one-position-at-a-time optimization protocol that utilizes metrics (such as shape complementarity) and local refinement methods too computationally expensive for global Monte Carlo (MC) sequence optimization. This protocol should be broadly useful for improving the stability and function of designed binding proteins. Proteins 2013. textcopyright 2013 Wiley Periodicals, Inc.}, keywords = {}, pubstate = {published}, tppubtype = {article} } In the design of new enzymes and binding proteins, human intuition is often used to modify computationally designed amino acid sequences prior to experimental characterization. The manual sequence changes involve both reversions of amino acid mutations back to the identity present in the parent scaffold and the introduction of residues making additional interactions with the binding partner or backing up first shell interactions. Automation of this manual sequence refinement process would allow more systematic evaluation and considerably reduce the amount of human designer effort involved. Here we introduce a benchmark for evaluating the ability of automated methods to recapitulate the sequence changes made to computer-generated models by human designers, and use it to assess alternative computational methods. We find the best performance for a greedy one-position-at-a-time optimization protocol that utilizes metrics (such as shape complementarity) and local refinement methods too computationally expensive for global Monte Carlo (MC) sequence optimization. This protocol should be broadly useful for improving the stability and function of designed binding proteins. Proteins 2013. textcopyright 2013 Wiley Periodicals, Inc. |
Conway, Patrick; Tyka, Michael D; DiMaio, Frank; Konerding, David E; Baker, David Relaxation of backbone bond geometry improves protein energy landscape modeling. Journal Article Protein science : a publication of the Protein Society, 2013, ISSN: 1469-896X. @article{514, title = {Relaxation of backbone bond geometry improves protein energy landscape modeling.}, author = { Patrick Conway and Michael D Tyka and Frank DiMaio and David E Konerding and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Conway_ProteinScience_2013.pdf}, doi = {10.1002/pro.2389}, issn = {1469-896X}, year = {2013}, date = {2013-10-01}, journal = {Protein science : a publication of the Protein Society}, abstract = {A key issue in macromolecular structure modeling is the granularity of the molecular representation. A fine-grained representation can approximate the actual structure more accurately, but may require many more degrees of freedom than a coarse-grained representation and hence make conformational search more challenging. We investigate this tradeoff between the accuracy and the size of protein conformational search space for two frequently used representations: one with fixed bond angles and lengths and one that has full flexibility. We performed large-scale explorations of the energy landscapes of 82 protein domains under each model, and find that the introduction of bond angle flexibility significantly increases the average energy gap between native and non-native structures. We also find that incorporating bonded geometry flexibility improves low resolution X-ray crystallographic refinement. These results suggest that backbone bond angle relaxation makes an important contribution to native structure energetics, that current energy functions are sufficiently accurate to capture the energetic gain associated with subtle deformations from chain ideality, and more speculatively, that backbone geometry distortions occur late in protein folding to optimize packing in the native state.}, keywords = {}, pubstate = {published}, tppubtype = {article} } A key issue in macromolecular structure modeling is the granularity of the molecular representation. A fine-grained representation can approximate the actual structure more accurately, but may require many more degrees of freedom than a coarse-grained representation and hence make conformational search more challenging. We investigate this tradeoff between the accuracy and the size of protein conformational search space for two frequently used representations: one with fixed bond angles and lengths and one that has full flexibility. We performed large-scale explorations of the energy landscapes of 82 protein domains under each model, and find that the introduction of bond angle flexibility significantly increases the average energy gap between native and non-native structures. We also find that incorporating bonded geometry flexibility improves low resolution X-ray crystallographic refinement. These results suggest that backbone bond angle relaxation makes an important contribution to native structure energetics, that current energy functions are sufficiently accurate to capture the energetic gain associated with subtle deformations from chain ideality, and more speculatively, that backbone geometry distortions occur late in protein folding to optimize packing in the native state. |
Bjelic, Sinisa; Kipnis, Yakov; Wang, Ling; Pianowski, Zbigniew; Vorobiev, Sergey; Su, Min; Seetharaman, Jayaraman; Xiao, Rong; Kornhaber, Gregory; Hunt, John F; Tong, Liang; Hilvert, Donald; Baker, David Exploration of Alternate Catalytic Mechanisms and Optimization Strategies for Retroaldolase Design Journal Article Journal of molecular biology, 426 , pp. 256-271, 2013, ISSN: 1089-8638. @article{494, title = {Exploration of Alternate Catalytic Mechanisms and Optimization Strategies for Retroaldolase Design}, author = { Sinisa Bjelic and Yakov Kipnis and Ling Wang and Zbigniew Pianowski and Sergey Vorobiev and Min Su and Jayaraman Seetharaman and Rong Xiao and Gregory Kornhaber and John F Hunt and Liang Tong and Donald Hilvert and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Bjelic_JMB_13N.pdf}, doi = {10.1016/j.jmb.2013.10.012}, issn = {1089-8638}, year = {2013}, date = {2013-10-01}, journal = {Journal of molecular biology}, volume = {426}, pages = {256-271}, chapter = {256}, abstract = {Designed retroaldolases have utilized a nucleophilic lysine to promote carbon-carbon bond cleavage of β-hydroxy-ketones via a covalent Schiff base intermediate. Previous computational designs have incorporated a water molecule to facilitate formation and breakdown of the carbinolamine intermediate to give the Schiff base and to function as a general acid/base. Here we investigate an alternative active-site design in which the catalytic water molecule was replaced by the side chain of a glutamic acid. Five out of seven designs expressed solubly and exhibited catalytic efficiencies similar to previously designed retroaldolases for the conversion of 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone to 6-methoxy-2-naphthaldehyde and acetone. After one round of site-directed saturation mutagenesis, improved variants of the two best designs, RA114 and RA117, exhibited among the highest kcat (>10(-3)s(-1)) and kcat/KM (11-25M(-1)s(-1)) values observed for retroaldolase designs prior to comprehensive directed evolution. In both cases, the >10(5)-fold rate accelerations that were achieved are within 1-3 orders of magnitude of the rate enhancements reported for the best catalysts for related reactions, including catalytic antibodies (kcat/kuncat=10(6) to 10(8)) and an extensively evolved computational design (kcat/kuncat>10(7)). The catalytic sites, revealed by X-ray structures of optimized versions of the two active designs, are in close agreement with the design models except for the catalytic lysine in RA114. We further improved the variants by computational remodeling of the loops and yeast display selection for reactivity of the catalytic lysine with a diketone probe, obtaining an additional order of magnitude enhancement in activity with both approaches.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Designed retroaldolases have utilized a nucleophilic lysine to promote carbon-carbon bond cleavage of β-hydroxy-ketones via a covalent Schiff base intermediate. Previous computational designs have incorporated a water molecule to facilitate formation and breakdown of the carbinolamine intermediate to give the Schiff base and to function as a general acid/base. Here we investigate an alternative active-site design in which the catalytic water molecule was replaced by the side chain of a glutamic acid. Five out of seven designs expressed solubly and exhibited catalytic efficiencies similar to previously designed retroaldolases for the conversion of 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone to 6-methoxy-2-naphthaldehyde and acetone. After one round of site-directed saturation mutagenesis, improved variants of the two best designs, RA114 and RA117, exhibited among the highest kcat (>10(-3)s(-1)) and kcat/KM (11-25M(-1)s(-1)) values observed for retroaldolase designs prior to comprehensive directed evolution. In both cases, the >10(5)-fold rate accelerations that were achieved are within 1-3 orders of magnitude of the rate enhancements reported for the best catalysts for related reactions, including catalytic antibodies (kcat/kuncat=10(6) to 10(8)) and an extensively evolved computational design (kcat/kuncat>10(7)). The catalytic sites, revealed by X-ray structures of optimized versions of the two active designs, are in close agreement with the design models except for the catalytic lysine in RA114. We further improved the variants by computational remodeling of the loops and yeast display selection for reactivity of the catalytic lysine with a diketone probe, obtaining an additional order of magnitude enhancement in activity with both approaches. |
Vernon, Robert; Shen, Yang; Baker, David; Lange, Oliver F Improved chemical shift based fragment selection for CS-Rosetta using Rosetta3 fragment picker. Journal Article Journal of biomolecular NMR, 57 , pp. 117-27, 2013, ISSN: 1573-5001. @article{508, title = {Improved chemical shift based fragment selection for CS-Rosetta using Rosetta3 fragment picker.}, author = { Robert Vernon and Yang Shen and David Baker and Oliver F Lange}, doi = {10.1007/s10858-013-9772-4}, issn = {1573-5001}, year = {2013}, date = {2013-10-01}, journal = {Journal of biomolecular NMR}, volume = {57}, pages = {117-27}, abstract = {A new fragment picker has been developed for CS-Rosetta that combines beneficial features of the original fragment picker, MFR, used with CS-Rosetta, and the fragment picker, NNMake, that was used for purely sequence based fragment selection in the context of ROSETTA de-novo structure prediction. Additionally, the new fragment picker has reduced sensitivity to outliers and other difficult to match data points rendering the protocol more robust and less likely to introduce bias towards wrong conformations in cases where data is bad, missing or inconclusive. The fragment picker protocol gives significant improvements on 6 of 23 CS-Rosetta targets. An independent benchmark on 39 protein targets, whose NMR data sets were published only after protocol optimization had been finished, also show significantly improved performance for the new fragment picker (van der Schot et al. in J Biomol NMR, 2013).}, keywords = {}, pubstate = {published}, tppubtype = {article} } A new fragment picker has been developed for CS-Rosetta that combines beneficial features of the original fragment picker, MFR, used with CS-Rosetta, and the fragment picker, NNMake, that was used for purely sequence based fragment selection in the context of ROSETTA de-novo structure prediction. Additionally, the new fragment picker has reduced sensitivity to outliers and other difficult to match data points rendering the protocol more robust and less likely to introduce bias towards wrong conformations in cases where data is bad, missing or inconclusive. The fragment picker protocol gives significant improvements on 6 of 23 CS-Rosetta targets. An independent benchmark on 39 protein targets, whose NMR data sets were published only after protocol optimization had been finished, also show significantly improved performance for the new fragment picker (van der Schot et al. in J Biomol NMR, 2013). |
Cooper, Seth; Khatib, Firas; Baker, David Increasing public involvement in structural biology Journal Article Structure (London, England : 1993), 21 , pp. 1482-4, 2013, ISSN: 1878-4186. @article{486, title = {Increasing public involvement in structural biology}, author = { Seth Cooper and Firas Khatib and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Cooper13L.pdf}, doi = {10.1016/j.str.2013.08.009}, issn = {1878-4186}, year = {2013}, date = {2013-09-01}, journal = {Structure (London, England : 1993)}, volume = {21}, pages = {1482-4}, abstract = {Public participation in scientific research can be a powerful supplement to more-traditional approaches. We~discuss aspects of the public participation project Foldit that may help others interested in starting their own projects.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Public participation in scientific research can be a powerful supplement to more-traditional approaches. We~discuss aspects of the public participation project Foldit that may help others interested in starting their own projects. |
Kamisetty, Hetunandan; Ovchinnikov, Sergey; Baker, David Assessing the utility of coevolution-based residue-residue contact predictions in a sequence- and structure-rich era. Journal Article Proceedings of the National Academy of Sciences of the United States of America, 110 , pp. 15674-9, 2013, ISSN: 1091-6490. @article{498, title = {Assessing the utility of coevolution-based residue-residue contact predictions in a sequence- and structure-rich era.}, author = { Hetunandan Kamisetty and Sergey Ovchinnikov and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Kamisetty_PNAS_2013.pdf}, doi = {10.1073/pnas.1314045110}, issn = {1091-6490}, year = {2013}, date = {2013-09-01}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {110}, pages = {15674-9}, abstract = {Recently developed methods have shown considerable promise in predicting residue-residue contacts in protein 3D structures using evolutionary covariance information. However, these methods require large numbers of evolutionarily related sequences to robustly assess the extent of residue covariation, and the larger the protein family, the more likely that contact information is unnecessary because a reasonable model can be built based on the structure of a homolog. Here we describe a method that integrates sequence coevolution and structural context information using a pseudolikelihood approach, allowing more accurate contact predictions from fewer homologous sequences. We rigorously assess the utility of predicted contacts for protein structure prediction using large and representative sequence and structure databases from recent structure prediction experiments. We find that contact predictions are likely to be accurate when the number of aligned sequences (with sequence redundancy reduced to 90%) is greater than five times the length of the protein, and that accurate predictions are likely to be useful for structure modeling if the aligned sequences are more similar to the protein of interest than to the closest homolog of known structure. These conditions are currently met by 422 of the protein families collected in the Pfam database.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Recently developed methods have shown considerable promise in predicting residue-residue contacts in protein 3D structures using evolutionary covariance information. However, these methods require large numbers of evolutionarily related sequences to robustly assess the extent of residue covariation, and the larger the protein family, the more likely that contact information is unnecessary because a reasonable model can be built based on the structure of a homolog. Here we describe a method that integrates sequence coevolution and structural context information using a pseudolikelihood approach, allowing more accurate contact predictions from fewer homologous sequences. We rigorously assess the utility of predicted contacts for protein structure prediction using large and representative sequence and structure databases from recent structure prediction experiments. We find that contact predictions are likely to be accurate when the number of aligned sequences (with sequence redundancy reduced to 90%) is greater than five times the length of the protein, and that accurate predictions are likely to be useful for structure modeling if the aligned sequences are more similar to the protein of interest than to the closest homolog of known structure. These conditions are currently met by 422 of the protein families collected in the Pfam database. |
Procko, Erik; Hedman, Rickard; Hamilton, Keith; Seetharaman, Jayaraman; Fleishman, Sarel J; Su, Min; Aramini, James; Kornhaber, Gregory; Hunt, John F; Tong, Liang; Montelione, Gaetano T; Baker, David Computational design of a protein-based enzyme inhibitor. Journal Article Journal of molecular biology, 425 , pp. 3563-75, 2013, ISSN: 1089-8638. @article{511, title = {Computational design of a protein-based enzyme inhibitor.}, author = { Erik Procko and Rickard Hedman and Keith Hamilton and Jayaraman Seetharaman and Sarel J Fleishman and Min Su and James Aramini and Gregory Kornhaber and John F Hunt and Liang Tong and Gaetano T Montelione and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Procko13.pdf}, doi = {10.1016/j.jmb.2013.06.035}, issn = {1089-8638}, year = {2013}, date = {2013-09-01}, journal = {Journal of molecular biology}, volume = {425}, pages = {3563-75}, abstract = {While there has been considerable progress in designing protein-protein interactions, the design of proteins that bind polar surfaces is an unmet challenge. We describe the computational design of a protein that binds the acidic active site of hen egg lysozyme and inhibits the enzyme. The design process starts with two polar amino acids that fit deep into the enzyme active site, identifies a protein scaffold that supports these residues and is complementary in shape to the lysozyme active-site region, and finally optimizes the surrounding contact surface for high-affinity binding. Following affinity maturation, a protein designed using this method bound lysozyme with low nanomolar affinity, and a combination of NMR studies, crystallography, and knockout mutagenesis confirmed the designed binding surface and orientation. Saturation mutagenesis with selection and deep sequencing demonstrated that specific designed interactions extending well beyond the centrally grafted polar residues are critical for high-affinity binding.}, keywords = {}, pubstate = {published}, tppubtype = {article} } While there has been considerable progress in designing protein-protein interactions, the design of proteins that bind polar surfaces is an unmet challenge. We describe the computational design of a protein that binds the acidic active site of hen egg lysozyme and inhibits the enzyme. The design process starts with two polar amino acids that fit deep into the enzyme active site, identifies a protein scaffold that supports these residues and is complementary in shape to the lysozyme active-site region, and finally optimizes the surrounding contact surface for high-affinity binding. Following affinity maturation, a protein designed using this method bound lysozyme with low nanomolar affinity, and a combination of NMR studies, crystallography, and knockout mutagenesis confirmed the designed binding surface and orientation. Saturation mutagenesis with selection and deep sequencing demonstrated that specific designed interactions extending well beyond the centrally grafted polar residues are critical for high-affinity binding. |
Tinberg, Christine E; Khare, Sagar D; Dou, Jiayi; Doyle, Lindsey; Nelson, Jorgen W; Schena, Alberto; Jankowski, Wojciech; Kalodimos, Charalampos G; Johnsson, Kai; Stoddard, Barry L; Baker, David Computational design of ligand-binding proteins with high affinity and selectivity Journal Article Nature, 501 , pp. 212-6, 2013, ISSN: 1476-4687. @article{480, title = {Computational design of ligand-binding proteins with high affinity and selectivity}, author = { Christine E Tinberg and Sagar D Khare and Jiayi Dou and Lindsey Doyle and Jorgen W Nelson and Alberto Schena and Wojciech Jankowski and Charalampos G Kalodimos and Kai Johnsson and Barry L Stoddard and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Tinberg13K.pdf}, doi = {10.1038/nature12443}, issn = {1476-4687}, year = {2013}, date = {2013-09-01}, journal = {Nature}, volume = {501}, pages = {212-6}, abstract = {The ability to design proteins with high affinity and selectivity for any given small molecule is a rigorous test of our understanding of the physiochemical principles that govern molecular recognition. Attempts to rationally design ligand-binding proteins have met with little success, however, and the computational design of protein-small-molecule interfaces remains an unsolved problem. Current approaches for designing ligand-binding proteins for medical and biotechnological uses rely on raising antibodies against a target antigen in immunized animals and/or performing laboratory-directed evolution of proteins with an existing low affinity for the desired ligand, neither of which allows complete control over the interactions involved in binding. Here we describe a general computational method for designing pre-organized and shape complementary small-molecule-binding sites, and use it to generate protein binders to the steroid digoxigenin (DIG). Of seventeen experimentally characterized designs, two bind DIG; the model of the higher affinity binder has the most energetically favourable and pre-organized interface in the design set. A comprehensive binding-fitness landscape of this design, generated by library selections and deep sequencing, was used to optimize its binding affinity to a picomolar level, and X-ray co-crystal structures of two variants show atomic-level agreement with the corresponding computational models. The optimized binder is selective for DIG over the related steroids digitoxigenin, progesterone and β-oestradiol, and this steroid binding preference can be reprogrammed by manipulation of explicitly designed hydrogen-bonding interactions. The computational design method presented here should enable the development of a new generation of biosensors, therapeutics and diagnostics.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The ability to design proteins with high affinity and selectivity for any given small molecule is a rigorous test of our understanding of the physiochemical principles that govern molecular recognition. Attempts to rationally design ligand-binding proteins have met with little success, however, and the computational design of protein-small-molecule interfaces remains an unsolved problem. Current approaches for designing ligand-binding proteins for medical and biotechnological uses rely on raising antibodies against a target antigen in immunized animals and/or performing laboratory-directed evolution of proteins with an existing low affinity for the desired ligand, neither of which allows complete control over the interactions involved in binding. Here we describe a general computational method for designing pre-organized and shape complementary small-molecule-binding sites, and use it to generate protein binders to the steroid digoxigenin (DIG). Of seventeen experimentally characterized designs, two bind DIG; the model of the higher affinity binder has the most energetically favourable and pre-organized interface in the design set. A comprehensive binding-fitness landscape of this design, generated by library selections and deep sequencing, was used to optimize its binding affinity to a picomolar level, and X-ray co-crystal structures of two variants show atomic-level agreement with the corresponding computational models. The optimized binder is selective for DIG over the related steroids digitoxigenin, progesterone and β-oestradiol, and this steroid binding preference can be reprogrammed by manipulation of explicitly designed hydrogen-bonding interactions. The computational design method presented here should enable the development of a new generation of biosensors, therapeutics and diagnostics. |
Mills, Jeremy H; Khare, Sagar D; Bolduc, Jill M; Forouhar, Farhad; Mulligan, Vikram Khipple; Lew, Scott; Seetharaman, Jayaraman; Tong, Liang; Stoddard, Barry L; Baker, David Computational design of an unnatural amino acid dependent metalloprotein with atomic level accuracy. Journal Article Journal of the American Chemical Society, 135 , pp. 13393-9, 2013, ISSN: 1520-5126. @article{509, title = {Computational design of an unnatural amino acid dependent metalloprotein with atomic level accuracy.}, author = { Jeremy H Mills and Sagar D Khare and Jill M Bolduc and Farhad Forouhar and Vikram Khipple Mulligan and Scott Lew and Jayaraman Seetharaman and Liang Tong and Barry L Stoddard and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Mills13.pdf}, doi = {10.1021/ja403503m}, issn = {1520-5126}, year = {2013}, date = {2013-09-01}, journal = {Journal of the American Chemical Society}, volume = {135}, pages = {13393-9}, abstract = {Genetically encoded unnatural amino acids could facilitate the design of proteins and enzymes of novel function, but correctly specifying sites of incorporation and the identities and orientations of surrounding residues represents a formidable challenge. Computational design methods have been used to identify optimal locations for functional sites in proteins and design the surrounding residues but have not incorporated unnatural amino acids in this process. We extended the Rosetta design methodology to design metalloproteins in which the amino acid (2,2textquoteright-bipyridin-5yl)alanine (Bpy-Ala) is a primary ligand of a bound metal ion. Following initial results that indicated the importance of buttressing the Bpy-Ala amino acid, we designed a buried metal binding site with octahedral coordination geometry consisting of Bpy-Ala, two protein-based metal ligands, and two metal-bound water molecules. Experimental characterization revealed a Bpy-Ala-mediated metalloprotein with the ability to bind divalent cations including Co(2+), Zn(2+), Fe(2+), and Ni(2+), with a Kd for Zn(2+) of ~40 pM. X-ray crystal structures of the designed protein bound to Co(2+) and Ni(2+) have RMSDs to the design model of 0.9 and 1.0 r A respectively over all atoms in the binding site.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Genetically encoded unnatural amino acids could facilitate the design of proteins and enzymes of novel function, but correctly specifying sites of incorporation and the identities and orientations of surrounding residues represents a formidable challenge. Computational design methods have been used to identify optimal locations for functional sites in proteins and design the surrounding residues but have not incorporated unnatural amino acids in this process. We extended the Rosetta design methodology to design metalloproteins in which the amino acid (2,2textquoteright-bipyridin-5yl)alanine (Bpy-Ala) is a primary ligand of a bound metal ion. Following initial results that indicated the importance of buttressing the Bpy-Ala amino acid, we designed a buried metal binding site with octahedral coordination geometry consisting of Bpy-Ala, two protein-based metal ligands, and two metal-bound water molecules. Experimental characterization revealed a Bpy-Ala-mediated metalloprotein with the ability to bind divalent cations including Co(2+), Zn(2+), Fe(2+), and Ni(2+), with a Kd for Zn(2+) of ~40 pM. X-ray crystal structures of the designed protein bound to Co(2+) and Ni(2+) have RMSDs to the design model of 0.9 and 1.0 r A respectively over all atoms in the binding site. |
van der Schot, Gijs; Zhang, Zaiyong; Vernon, Robert; Shen, Yang; Vranken, Wim F; Baker, David; Bonvin, Alexandre M J J; Lange, Oliver F Improving 3D structure prediction from chemical shift data. Journal Article Journal of biomolecular NMR, 57 , pp. 27-35, 2013, ISSN: 1573-5001. @article{507, title = {Improving 3D structure prediction from chemical shift data.}, author = { Gijs van der Schot and Zaiyong Zhang and Robert Vernon and Yang Shen and Wim F Vranken and David Baker and Alexandre M J J Bonvin and Oliver F Lange}, doi = {10.1007/s10858-013-9762-6}, issn = {1573-5001}, year = {2013}, date = {2013-09-01}, journal = {Journal of biomolecular NMR}, volume = {57}, pages = {27-35}, abstract = {We report advances in the calculation of protein structures from chemical shift nuclear magnetic resonance data alone. Our previously developed method, CS-Rosetta, assembles structures from a library of short protein fragments picked from a large library of protein structures using chemical shifts and sequence information. Here we demonstrate that combination of a new and improved fragment picker and the iterative sampling algorithm RASREC yield significant improvements in convergence and accuracy. Moreover, we introduce improved criteria for assessing the accuracy of the models produced by the method. The method was tested on 39 proteins in the 50-100 residue size range and yields reliable structures in 70~% of the cases. All structures that passed the reliability filter were accurate (<2~r A RMSD from the reference).}, keywords = {}, pubstate = {published}, tppubtype = {article} } We report advances in the calculation of protein structures from chemical shift nuclear magnetic resonance data alone. Our previously developed method, CS-Rosetta, assembles structures from a library of short protein fragments picked from a large library of protein structures using chemical shifts and sequence information. Here we demonstrate that combination of a new and improved fragment picker and the iterative sampling algorithm RASREC yield significant improvements in convergence and accuracy. Moreover, we introduce improved criteria for assessing the accuracy of the models produced by the method. The method was tested on 39 proteins in the 50-100 residue size range and yields reliable structures in 70~% of the cases. All structures that passed the reliability filter were accurate (<2~r A RMSD from the reference). |
Giger, Lars; Caner, Sami; Obexer, Richard; Kast, Peter; Baker, David; Ban, Nenad; Hilvert, Donald Evolution of a designed retro-aldolase leads to complete active site remodeling. Journal Article Nature chemical biology, 9 , pp. 494-8, 2013, ISSN: 1552-4469. @article{504, title = {Evolution of a designed retro-aldolase leads to complete active site remodeling.}, author = { Lars Giger and Sami Caner and Richard Obexer and Peter Kast and David Baker and Nenad Ban and Donald Hilvert}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Giger_nchembio_2013.pdf}, doi = {10.1038/nchembio.1276}, issn = {1552-4469}, year = {2013}, date = {2013-08-01}, journal = {Nature chemical biology}, volume = {9}, pages = {494-8}, abstract = {Evolutionary advances are often fueled by unanticipated innovation. Directed evolution of a computationally designed enzyme suggests that pronounced molecular changes can also drive the optimization of primitive protein active sites. The specific activity of an artificial retro-aldolase was boosted >4,400-fold by random mutagenesis and screening, affording catalytic efficiencies approaching those of natural enzymes. However, structural and mechanistic studies reveal that the engineered catalytic apparatus, consisting of a reactive lysine and an ordered water molecule, was unexpectedly abandoned in favor of a new lysine residue in a substrate-binding pocket created during the optimization process. Structures of the initial in silico design, a mechanistically promiscuous intermediate and one of the most evolved variants highlight the importance of loop mobility and supporting functional groups in the emergence of the new catalytic center. Such internal competition between alternative reactive sites may have characterized the early evolution of many natural enzymes.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Evolutionary advances are often fueled by unanticipated innovation. Directed evolution of a computationally designed enzyme suggests that pronounced molecular changes can also drive the optimization of primitive protein active sites. The specific activity of an artificial retro-aldolase was boosted >4,400-fold by random mutagenesis and screening, affording catalytic efficiencies approaching those of natural enzymes. However, structural and mechanistic studies reveal that the engineered catalytic apparatus, consisting of a reactive lysine and an ordered water molecule, was unexpectedly abandoned in favor of a new lysine residue in a substrate-binding pocket created during the optimization process. Structures of the initial in silico design, a mechanistically promiscuous intermediate and one of the most evolved variants highlight the importance of loop mobility and supporting functional groups in the emergence of the new catalytic center. Such internal competition between alternative reactive sites may have characterized the early evolution of many natural enzymes. |
Kim, David E; DiMaio, Frank; Wang, Ray Yu-Ruei; Song, Yifan; Baker, David One contact for every twelve residues allows robust and accurate topology-level protein structure modeling. Journal Article Proteins, 2013, ISSN: 1097-0134. @article{506, title = {One contact for every twelve residues allows robust and accurate topology-level protein structure modeling.}, author = { David E Kim and Frank DiMaio and Ray Yu-Ruei Wang and Yifan Song and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Kim_Proteins_2013.pdf}, doi = {10.1002/prot.24374}, issn = {1097-0134}, year = {2013}, date = {2013-07-01}, journal = {Proteins}, abstract = {A number of methods have been described for identifying pairs of contacting residues in protein three-dimensional structures, but it is unclear how many contacts are required for accurate structure modeling. The CASP10 assisted contact experiment provided a blind test of contact guided protein structure modeling. We describe the models generated for these contact guided prediction challenges using the Rosetta structure modeling methodology. For nearly all cases, the submitted models had the correct overall topology, and in some cases, they had near atomic-level accuracy; for example the model of the 384 residue homo-oligomeric tetramer (Tc680o) had only 2.9 r A root-mean-square deviation (RMSD) from the crystal structure. Our results suggest that experimental and bioinformatic methods for obtaining contact information may need to generate only one correct contact for every 12 residues in the protein to allow accurate topology level modeling. Proteins 2013;. textcopyright 2013 Wiley Periodicals, Inc.}, keywords = {}, pubstate = {published}, tppubtype = {article} } A number of methods have been described for identifying pairs of contacting residues in protein three-dimensional structures, but it is unclear how many contacts are required for accurate structure modeling. The CASP10 assisted contact experiment provided a blind test of contact guided protein structure modeling. We describe the models generated for these contact guided prediction challenges using the Rosetta structure modeling methodology. For nearly all cases, the submitted models had the correct overall topology, and in some cases, they had near atomic-level accuracy; for example the model of the 384 residue homo-oligomeric tetramer (Tc680o) had only 2.9 r A root-mean-square deviation (RMSD) from the crystal structure. Our results suggest that experimental and bioinformatic methods for obtaining contact information may need to generate only one correct contact for every 12 residues in the protein to allow accurate topology level modeling. Proteins 2013;. textcopyright 2013 Wiley Periodicals, Inc. |