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2015 |
Goldsmith, M; Eckstein, S; Ashani, Y; Greisen, Jr P; Leader, H; Sussman, JL; Aggarwal, N; Ovchinnikov, S; Tawfik, DS; Baker, D; Thiermann, H; Worek, F Catalytic efficiencies of directly evolved phosphotriesterase variants with structurally different organophosphorus compounds in vitro Journal Article Archives of Toxicology, 2015. @article{M2015, title = {Catalytic efficiencies of directly evolved phosphotriesterase variants with structurally different organophosphorus compounds in vitro}, author = {M Goldsmith and S Eckstein and Y Ashani and P Jr Greisen and H Leader and JL Sussman and N Aggarwal and S Ovchinnikov and DS Tawfik and D Baker and H Thiermann and F Worek}, url = {https://www.bakerlab.org/wp-content/uploads/2015/12/Goldsmith_ArchToxicol_2015.pdf}, doi = {10.1007/s00204-015-1626-2}, year = {2015}, date = {2015-11-26}, journal = {Archives of Toxicology}, abstract = {The nearly 200,000 fatalities following exposure to organophosphorus (OP) pesticides each year and the omnipresent danger of a terroristic attack with OP nerve agents emphasize the demand for the development of effective OP antidotes. Standard treatments for intoxicated patients with a combination of atropine and an oxime are limited in their efficacy. Thus, research focuses on developing catalytic bioscavengers as an alternative approach using OP-hydrolyzing enzymes such as Brevundimonas diminuta phosphotriesterase (PTE). Recently, a PTE mutant dubbed C23 was engineered, exhibiting reversed stereoselectivity and high catalytic efficiency (k cat/K M) for the hydrolysis of the toxic enantiomers of VX, CVX, and VR. Additionally, C23's ability to prevent systemic toxicity of VX using a low protein dose has been shown in vivo. In this study, the catalytic efficiencies of V-agent hydrolysis by two newly selected PTE variants were determined. Moreover, in order to establish trends in sequence-activity relationships along the pathway of PTE's laboratory evolution, we examined k cat/K M values of several variants with a number of V-type and G-type nerve agents as well as with different OP pesticides. Although none of the new PTE variants exhibited k cat/K M values >107 M-1 min-1 with V-type nerve agents, which is required for effective prophylaxis, they were improved with VR relative to previously evolved variants. The new variants detoxify a broad spectrum of OPs and provide insight into OP hydrolysis and sequence-activity relationships.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The nearly 200,000 fatalities following exposure to organophosphorus (OP) pesticides each year and the omnipresent danger of a terroristic attack with OP nerve agents emphasize the demand for the development of effective OP antidotes. Standard treatments for intoxicated patients with a combination of atropine and an oxime are limited in their efficacy. Thus, research focuses on developing catalytic bioscavengers as an alternative approach using OP-hydrolyzing enzymes such as Brevundimonas diminuta phosphotriesterase (PTE). Recently, a PTE mutant dubbed C23 was engineered, exhibiting reversed stereoselectivity and high catalytic efficiency (k cat/K M) for the hydrolysis of the toxic enantiomers of VX, CVX, and VR. Additionally, C23's ability to prevent systemic toxicity of VX using a low protein dose has been shown in vivo. In this study, the catalytic efficiencies of V-agent hydrolysis by two newly selected PTE variants were determined. Moreover, in order to establish trends in sequence-activity relationships along the pathway of PTE's laboratory evolution, we examined k cat/K M values of several variants with a number of V-type and G-type nerve agents as well as with different OP pesticides. Although none of the new PTE variants exhibited k cat/K M values >107 M-1 min-1 with V-type nerve agents, which is required for effective prophylaxis, they were improved with VR relative to previously evolved variants. The new variants detoxify a broad spectrum of OPs and provide insight into OP hydrolysis and sequence-activity relationships. |
Huang, PS; Feldmeier, K; Parmeggiani, F; Velasco, DA Fernandez; Höcker, B; Baker, D De novo design of a four-fold symmetric TIM-barrel protein with atomic-level accuracy Journal Article Nature Chemical Biology, 12(1) , pp. 29-34, 2015. @article{PS2015, title = {De novo design of a four-fold symmetric TIM-barrel protein with atomic-level accuracy}, author = {PS Huang and K Feldmeier and F Parmeggiani and DA Fernandez Velasco and B Höcker and D Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2015/12/Huang_NatChemBio_2015.pdf}, doi = {10.1038/nchembio.1966}, year = {2015}, date = {2015-11-23}, journal = {Nature Chemical Biology}, volume = {12(1)}, pages = {29-34}, abstract = {Despite efforts for over 25 years, de novo protein design has not succeeded in achieving the TIM-barrel fold. Here we describe the computational design of four-fold symmetrical (β/α)8 barrels guided by geometrical and chemical principles. Experimental characterization of 33 designs revealed the importance of side chain-backbone hydrogen bonds for defining the strand register between repeat units. The X-ray crystal structure of a designed thermostable 184-residue protein is nearly identical to that of the designed TIM-barrel model. PSI-BLAST searches do not identify sequence similarities to known TIM-barrel proteins, and sensitive profile-profile searches indicate that the design sequence is distant from other naturally occurring TIM-barrel superfamilies, suggesting that Nature has sampled only a subset of the sequence space available to the TIM-barrel fold. The ability to design TIM barrels de novo opens new possibilities for custom-made enzymes. }, keywords = {}, pubstate = {published}, tppubtype = {article} } Despite efforts for over 25 years, de novo protein design has not succeeded in achieving the TIM-barrel fold. Here we describe the computational design of four-fold symmetrical (β/α)8 barrels guided by geometrical and chemical principles. Experimental characterization of 33 designs revealed the importance of side chain-backbone hydrogen bonds for defining the strand register between repeat units. The X-ray crystal structure of a designed thermostable 184-residue protein is nearly identical to that of the designed TIM-barrel model. PSI-BLAST searches do not identify sequence similarities to known TIM-barrel proteins, and sensitive profile-profile searches indicate that the design sequence is distant from other naturally occurring TIM-barrel superfamilies, suggesting that Nature has sampled only a subset of the sequence space available to the TIM-barrel fold. The ability to design TIM barrels de novo opens new possibilities for custom-made enzymes. |
Lin YR Koga N, Tatsumi-Koga Liu Clouser AF Montelione GT Baker R G D Control over overall shape and size in de novo designed proteins Journal Article Proc Natl Acad Sci U S A., pp. E5478-85, 2015. @article{YR2015, title = {Control over overall shape and size in de novo designed proteins}, author = {Lin YR, Koga N, Tatsumi-Koga R, Liu G, Clouser AF, Montelione GT, Baker D}, url = {https://www.bakerlab.org/wp-content/uploads/2016/04/PNAS-2015-Lin-E5478-85.pdf}, doi = {10.1073/pnas.1509508112}, year = {2015}, date = {2015-10-06}, journal = {Proc Natl Acad Sci U S A.}, pages = {E5478-85}, abstract = {We recently described general principles for designing ideal protein structures stabilized by completely consistent local and nonlocal interactions. The principles relate secondary structure patterns to tertiary packing motifs and enable design of different protein topologies. To achieve fine control over protein shape and size within a particular topology, we have extended the design rules by systematically analyzing the codependencies between the lengths and packing geometry of successive secondary structure elements and the backbone torsion angles of the loop linking them. We demonstrate the control afforded by the resulting extended rule set by designing a series of proteins with the same fold but considerable variation in secondary structure length, loop geometry, β-strand registry, and overall shape. Solution NMR structures of four designed proteins for two different folds show that protein shape and size can be precisely controlled within a given protein fold. These extended design principles provide the foundation for custom design of protein structures performing desired functions. }, keywords = {}, pubstate = {published}, tppubtype = {article} } We recently described general principles for designing ideal protein structures stabilized by completely consistent local and nonlocal interactions. The principles relate secondary structure patterns to tertiary packing motifs and enable design of different protein topologies. To achieve fine control over protein shape and size within a particular topology, we have extended the design rules by systematically analyzing the codependencies between the lengths and packing geometry of successive secondary structure elements and the backbone torsion angles of the loop linking them. We demonstrate the control afforded by the resulting extended rule set by designing a series of proteins with the same fold but considerable variation in secondary structure length, loop geometry, β-strand registry, and overall shape. Solution NMR structures of four designed proteins for two different folds show that protein shape and size can be precisely controlled within a given protein fold. These extended design principles provide the foundation for custom design of protein structures performing desired functions. |
Holstein, Carly A; Chevalier, Aaron; Bennett, Steven; Anderson, Caitlin E; Keniston, Karen; Olsen, Cathryn; Li, Bing; Bales, Brian; Moore, David R; Fu, Elain; Baker, David; Yager, Paul Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers. Journal Article Analytical and bioanalytical chemistry, 2015, ISSN: 1618-2650. @article{626, title = {Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers.}, author = { Carly A Holstein and Aaron Chevalier and Steven Bennett and Caitlin E Anderson and Karen Keniston and Cathryn Olsen and Bing Li and Brian Bales and David R Moore and Elain Fu and David Baker and Paul Yager}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Holstien_Anal_Bioanal_Chem_2015.pdf}, doi = {10.1007/s00216-015-9052-0}, issn = {1618-2650}, year = {2015}, date = {2015-10-01}, journal = {Analytical and bioanalytical chemistry}, abstract = {To enable enhanced paper-based diagnostics with improved detection capabilities, new methods are needed to immobilize affinity reagents to porous substrates, especially for capture molecules other than IgG. To this end, we have developed and characterized three novel methods for immobilizing protein-based affinity reagents to nitrocellulose membranes. We have demonstrated these methods using recombinant affinity proteins for the influenza surface protein hemagglutinin, leveraging the customizability of these recombinant "flu binders" for the design of features for immobilization. The three approaches shown are: (1) covalent attachment of thiolated affinity protein to an epoxide-functionalized nitrocellulose membrane, (2) attachment of biotinylated affinity protein through a nitrocellulose-binding streptavidin anchor protein, and (3) fusion of affinity protein to a novel nitrocellulose-binding anchor protein for direct coupling and immobilization. We also characterized the use of direct adsorption for the flu binders, as a point of comparison and motivation for these novel methods. Finally, we demonstrated that these novel methods can provide improved performance to an influenza hemagglutinin assay, compared to a traditional antibody-based capture system. Taken together, this work advances the toolkit available for the development of next-generation paper-based diagnostics.}, keywords = {}, pubstate = {published}, tppubtype = {article} } To enable enhanced paper-based diagnostics with improved detection capabilities, new methods are needed to immobilize affinity reagents to porous substrates, especially for capture molecules other than IgG. To this end, we have developed and characterized three novel methods for immobilizing protein-based affinity reagents to nitrocellulose membranes. We have demonstrated these methods using recombinant affinity proteins for the influenza surface protein hemagglutinin, leveraging the customizability of these recombinant "flu binders" for the design of features for immobilization. The three approaches shown are: (1) covalent attachment of thiolated affinity protein to an epoxide-functionalized nitrocellulose membrane, (2) attachment of biotinylated affinity protein through a nitrocellulose-binding streptavidin anchor protein, and (3) fusion of affinity protein to a novel nitrocellulose-binding anchor protein for direct coupling and immobilization. We also characterized the use of direct adsorption for the flu binders, as a point of comparison and motivation for these novel methods. Finally, we demonstrated that these novel methods can provide improved performance to an influenza hemagglutinin assay, compared to a traditional antibody-based capture system. Taken together, this work advances the toolkit available for the development of next-generation paper-based diagnostics. |
S Ovchinnikov L Kinch, Park Liao Pei DE Kim Kamisetty NV Grishin Baker H Y J H D Large-scale determination of previously unsolved protein structures using evolutionary information Journal Article eLife, 2015. @article{S2015b, title = {Large-scale determination of previously unsolved protein structures using evolutionary information}, author = {S Ovchinnikov, L Kinch, H Park, Y Liao, J Pei, DE Kim, H Kamisetty, NV Grishin, D Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/01/Ovchinnikov_eLife_2015.pdf}, doi = {10.7554/eLife.09248}, year = {2015}, date = {2015-09-03}, journal = {eLife}, abstract = {The prediction of the structures of proteins without detectable sequence similarity to any protein of known structure remains an outstanding scientific challenge. Here we report significant progress in this area. We first describe de novo blind structure predictions of unprecendented accuracy we made for two proteins in large families in the recent CASP11 blind test of protein structure prediction methods by incorporating residue-residue co-evolution information in the Rosetta structure prediction program. We then describe the use of this method to generate structure models for 58 of the 121 large protein families in prokaryotes for which three-dimensional structures are not available. These models, which are posted online for public access, provide structural information for the over 400,000 proteins belonging to the 58 families and suggest hypotheses about mechanism for the subset for which the function is known, and hypotheses about function for the remainder. }, keywords = {}, pubstate = {published}, tppubtype = {article} } The prediction of the structures of proteins without detectable sequence similarity to any protein of known structure remains an outstanding scientific challenge. Here we report significant progress in this area. We first describe de novo blind structure predictions of unprecendented accuracy we made for two proteins in large families in the recent CASP11 blind test of protein structure prediction methods by incorporating residue-residue co-evolution information in the Rosetta structure prediction program. We then describe the use of this method to generate structure models for 58 of the 121 large protein families in prokaryotes for which three-dimensional structures are not available. These models, which are posted online for public access, provide structural information for the over 400,000 proteins belonging to the 58 families and suggest hypotheses about mechanism for the subset for which the function is known, and hypotheses about function for the remainder. |
Wolf, Clancey; Siegel, Justin B; Tinberg, Christine; Camarca, Alessandra; Gianfrani, Carmen; Paski, Shirley; Guan, Rongjin; Montelione, Gaetano T; Baker, David; Pultz, Ingrid S Engineering of Kuma030: a gliadin peptidase that rapidly degrades immunogenic gliadin peptides in gastric conditions. Journal Article Journal of the American Chemical Society, 2015, ISSN: 1520-5126. @article{617, title = {Engineering of Kuma030: a gliadin peptidase that rapidly degrades immunogenic gliadin peptides in gastric conditions.}, author = { Clancey Wolf and Justin B Siegel and Christine Tinberg and Alessandra Camarca and Carmen Gianfrani and Shirley Paski and Rongjin Guan and Gaetano T Montelione and David Baker and Ingrid S Pultz}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Wolf_JACS_2015.pdf}, doi = {10.1021/jacs.5b08325}, issn = {1520-5126}, year = {2015}, date = {2015-09-01}, journal = {Journal of the American Chemical Society}, abstract = {Celiac disease is characterized by intestinal inflammation triggered by gliadin, a component of dietary gluten. Oral administration of proteases that can rapidly degrade gliadin in the gastric compartment has been proposed as a treatment for celiac disease; however, no protease has been shown to specifically reduce the immunogenic gliadin content, in gastric conditions, to below the threshold shown to be toxic for celiac patients. Here, we used the Rosetta Molecular Modeling Suite to redesign the active site of the acid-active gliadin endopeptidase KumaMax. The resulting protease, Kuma030, specifically recognizes tripeptide sequences that are found throughout the immunogenic regions of gliadin, as well as in homologous proteins in barley and rye. Indeed, treatment of gliadin with Kuma030 eliminates the ability of gliadin to stimulate a T cell response. Kuma030 is capable of degrading >99% of the immunogenic gliadin fraction in laboratory-simulated gastric digestions with minutes, to a level below the toxic threshold for celiac patients, suggesting great potential for this enzyme as an oral therapeutic for celiac disease.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Celiac disease is characterized by intestinal inflammation triggered by gliadin, a component of dietary gluten. Oral administration of proteases that can rapidly degrade gliadin in the gastric compartment has been proposed as a treatment for celiac disease; however, no protease has been shown to specifically reduce the immunogenic gliadin content, in gastric conditions, to below the threshold shown to be toxic for celiac patients. Here, we used the Rosetta Molecular Modeling Suite to redesign the active site of the acid-active gliadin endopeptidase KumaMax. The resulting protease, Kuma030, specifically recognizes tripeptide sequences that are found throughout the immunogenic regions of gliadin, as well as in homologous proteins in barley and rye. Indeed, treatment of gliadin with Kuma030 eliminates the ability of gliadin to stimulate a T cell response. Kuma030 is capable of degrading >99% of the immunogenic gliadin fraction in laboratory-simulated gastric digestions with minutes, to a level below the toxic threshold for celiac patients, suggesting great potential for this enzyme as an oral therapeutic for celiac disease. |
Bale, Jacob B; Park, Rachel U; Liu, Yuxi; Gonen, Shane; Gonen, Tamir; Cascio, Duilio; King, Neil P; Yeates, Todd O; Baker, David Structure of a designed tetrahedral protein assembly variant engineered to have improved soluble expression Journal Article Protein science : a publication of the Protein Society, 2015, ISSN: 1469-896X. @article{616, title = {Structure of a designed tetrahedral protein assembly variant engineered to have improved soluble expression}, author = { Jacob B Bale and Rachel U Park and Yuxi Liu and Shane Gonen and Tamir Gonen and Duilio Cascio and Neil P. King and Todd O. Yeates and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Bale_designed_tetrahedral_ProteinSci2015.pdf}, doi = {10.1002/pro.2748}, issn = {1469-896X}, year = {2015}, date = {2015-07-01}, journal = {Protein science : a publication of the Protein Society}, abstract = {We recently reported the development of a computational method for the design of coassembling multicomponent protein nanomaterials. While four such materials were validated at high-resolution by X-ray crystallography, low yield of soluble protein prevented X-ray structure determination of a fifth designed material, T33-09. Here we report the design and crystal structure of T33-31, a variant of T33-09 with improved soluble yield resulting from redesign efforts focused on mutating solvent-exposed side chains to charged amino acids. The structure is found to match the computational design model with atomic-level accuracy, providing further validation of the design approach and demonstrating a simple and potentially general means of improving the yield of designed protein nanomaterials.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We recently reported the development of a computational method for the design of coassembling multicomponent protein nanomaterials. While four such materials were validated at high-resolution by X-ray crystallography, low yield of soluble protein prevented X-ray structure determination of a fifth designed material, T33-09. Here we report the design and crystal structure of T33-31, a variant of T33-09 with improved soluble yield resulting from redesign efforts focused on mutating solvent-exposed side chains to charged amino acids. The structure is found to match the computational design model with atomic-level accuracy, providing further validation of the design approach and demonstrating a simple and potentially general means of improving the yield of designed protein nanomaterials. |
Gonen, Shane; DiMaio, Frank; Gonen, Tamir; Baker, David Design of ordered two-dimensional arrays mediated by noncovalent protein-protein interfaces Journal Article Science (New York, N.Y.), 348 , pp. 1365-8, 2015, ISSN: 1095-9203. @article{613, title = {Design of ordered two-dimensional arrays mediated by noncovalent protein-protein interfaces}, author = { Shane Gonen and Frank DiMaio and Tamir Gonen and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Gonen_2DArrays_Baker2015.pdf}, doi = {10.1126/science.aaa9897}, issn = {1095-9203}, year = {2015}, date = {2015-06-01}, journal = {Science (New York, N.Y.)}, volume = {348}, pages = {1365-8}, abstract = {We describe a general approach to designing two-dimensional (2D) protein arrays mediated by noncovalent protein-protein interfaces. Protein homo-oligomers are placed into one of the seventeen 2D layer groups, the degrees of freedom of the lattice are sampled to identify configurations with shape-complementary interacting surfaces, and the interaction energy is minimized using sequence design calculations. We used the method to design proteins that self-assemble into layer groups P 3 2 1, P 4 2(1) 2, and P 6. Projection maps of micrometer-scale arrays, assembled both in vitro and in vivo, are consistent with the design models and display the target layer group symmetry. Such programmable 2D protein lattices should enable new approaches to structure determination, sensing, and nanomaterial engineering.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We describe a general approach to designing two-dimensional (2D) protein arrays mediated by noncovalent protein-protein interfaces. Protein homo-oligomers are placed into one of the seventeen 2D layer groups, the degrees of freedom of the lattice are sampled to identify configurations with shape-complementary interacting surfaces, and the interaction energy is minimized using sequence design calculations. We used the method to design proteins that self-assemble into layer groups P 3 2 1, P 4 2(1) 2, and P 6. Projection maps of micrometer-scale arrays, assembled both in vitro and in vivo, are consistent with the design models and display the target layer group symmetry. Such programmable 2D protein lattices should enable new approaches to structure determination, sensing, and nanomaterial engineering. |
Park, Hahnbeom; DiMaio, Frank; Baker, David The origin of consistent protein structure refinement from structural averaging. Journal Article Structure (London, England : 1993), 23 , pp. 1123-8, 2015, ISSN: 1878-4186. @article{615, title = {The origin of consistent protein structure refinement from structural averaging.}, author = { Hahnbeom Park and Frank DiMaio and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Park_Structure_2015.pdf http://www.ncbi.nlm.nih.gov/pubmed/?term=The+Origin+of+Consistent+Protein+Structure+Refinement+from+Structural+Averaging}, doi = {10.1016/j.str.2015.03.022}, issn = {1878-4186}, year = {2015}, date = {2015-06-01}, journal = {Structure (London, England : 1993)}, volume = {23}, pages = {1123-8}, abstract = {Recent studies have shown that explicit solvent molecular dynamics (MD) simulation followed by structural averaging can consistently improve protein structure models. We find that improvement upon averaging is not limited to explicit water MD simulation, as consistent improvements are also observed for more efficient implicit solvent MD or Monte Carlo minimization simulations. To determine the origin of these improvements, we examine the changes in model accuracy brought about by averaging at the individual residue level. We find that the improvement in model quality from averaging results from the superposition of two effects: a dampening of deviations from the correct structure in the least well modeled regions, and a reinforcement of consistent movements towards the correct structure in better modeled regions. These observations are consistent with an energy landscape model in which the magnitude of the energy gradient toward the native structure decreases with increasing distance from the native state.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Recent studies have shown that explicit solvent molecular dynamics (MD) simulation followed by structural averaging can consistently improve protein structure models. We find that improvement upon averaging is not limited to explicit water MD simulation, as consistent improvements are also observed for more efficient implicit solvent MD or Monte Carlo minimization simulations. To determine the origin of these improvements, we examine the changes in model accuracy brought about by averaging at the individual residue level. We find that the improvement in model quality from averaging results from the superposition of two effects: a dampening of deviations from the correct structure in the least well modeled regions, and a reinforcement of consistent movements towards the correct structure in better modeled regions. These observations are consistent with an energy landscape model in which the magnitude of the energy gradient toward the native structure decreases with increasing distance from the native state. |
Siegel, Justin B; Smith, Amanda Lee; Poust, Sean; Wargacki, Adam J; Bar-Even, Arren; Louw, Catherine; Shen, Betty W; Eiben, Christopher B; Tran, Huu M; Noor, Elad; Gallaher, Jasmine L; Bale, Jacob; Yoshikuni, Yasuo; Gelb, Michael H; Keasling, Jay D; Stoddard, Barry L; Lidstrom, Mary E; Baker, David Computational protein design enables a novel one-carbon assimilation pathway Journal Article Proceedings of the National Academy of Sciences of the United States of America, 2015, ISSN: 1091-6490. @article{565, title = {Computational protein design enables a novel one-carbon assimilation pathway}, author = { Justin B Siegel and Amanda Lee Smith and Sean Poust and Adam J Wargacki and Arren Bar-Even and Catherine Louw and Betty W Shen and Christopher B Eiben and Huu M Tran and Elad Noor and Jasmine L Gallaher and Jacob Bale and Yasuo Yoshikuni and Michael H Gelb and Jay D Keasling and Barry L Stoddard and Mary E Lidstrom and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/siegel15A.pdf}, doi = {10.1073/pnas.1500545112}, issn = {1091-6490}, year = {2015}, date = {2015-03-01}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, abstract = {We describe a computationally designed enzyme, formolase (FLS), which catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. The existence of FLS enables the design of a new carbon fixation pathway, the formolase pathway, consisting of a small number of thermodynamically favorable chemical transformations that convert formate into a three-carbon sugar in central metabolism. The formolase pathway is predicted to use carbon more efficiently and with less backward flux than any naturally occurring one-carbon assimilation pathway. When supplemented with enzymes carrying out the other steps in the pathway, FLS converts formate into dihydroxyacetone phosphate and other central metabolites in vitro. These results demonstrate how modern protein engineering and design tools can facilitate the construction of a completely new biosynthetic pathway.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We describe a computationally designed enzyme, formolase (FLS), which catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. The existence of FLS enables the design of a new carbon fixation pathway, the formolase pathway, consisting of a small number of thermodynamically favorable chemical transformations that convert formate into a three-carbon sugar in central metabolism. The formolase pathway is predicted to use carbon more efficiently and with less backward flux than any naturally occurring one-carbon assimilation pathway. When supplemented with enzymes carrying out the other steps in the pathway, FLS converts formate into dihydroxyacetone phosphate and other central metabolites in vitro. These results demonstrate how modern protein engineering and design tools can facilitate the construction of a completely new biosynthetic pathway. |
DiMaio, Frank; Song, Yifan; Li, Xueming; Brunner, Matthias J; Xu, Chunfu; Conticello, Vincent; Egelman, Edward; Marlovits, Thomas C; Cheng, Yifan; Baker, David Atomic-accuracy models from 4.5-r A cryo-electron microscopy data with density-guided iterative local refinement. Journal Article Nature methods, 2015, ISSN: 1548-7105. @article{560, title = {Atomic-accuracy models from 4.5-r A cryo-electron microscopy data with density-guided iterative local refinement.}, author = { Frank DiMaio and Yifan Song and Xueming Li and Matthias J Brunner and Chunfu Xu and Vincent Conticello and Edward Egelman and Thomas C Marlovits and Yifan Cheng and David Baker}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/DiMaio_NatMethods_2015.pdf}, doi = {10.1038/nmeth.3286}, issn = {1548-7105}, year = {2015}, date = {2015-02-01}, journal = {Nature methods}, abstract = {We describe a general approach for refining protein structure models on the basis of cryo-electron microscopy maps with near-atomic resolution. The method integrates Monte Carlo sampling with local density-guided optimization, Rosetta all-atom refinement and real-space B-factor fitting. In tests on experimental maps of three different systems with 4.5-r A resolution or better, the method consistently produced models with atomic-level accuracy largely independently of starting-model quality, and it outperformed the molecular dynamics-based MDFF method. Cross-validated model quality statistics correlated with model accuracy over the three test systems.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We describe a general approach for refining protein structure models on the basis of cryo-electron microscopy maps with near-atomic resolution. The method integrates Monte Carlo sampling with local density-guided optimization, Rosetta all-atom refinement and real-space B-factor fitting. In tests on experimental maps of three different systems with 4.5-r A resolution or better, the method consistently produced models with atomic-level accuracy largely independently of starting-model quality, and it outperformed the molecular dynamics-based MDFF method. Cross-validated model quality statistics correlated with model accuracy over the three test systems. |
Wang, Ray Yu-Ruei; Kudryashev, Mikhail; Li, Xueming; Egelman, Edward H; Basler, Marek; Cheng, Yifan; Baker, David; DiMaio, Frank De novo protein structure determination from near-atomic-resolution cryo-EM maps. Journal Article Nature methods, 2015, ISSN: 1548-7105. @article{559, title = {De novo protein structure determination from near-atomic-resolution cryo-EM maps.}, author = { Ray Yu-Ruei Wang and Mikhail Kudryashev and Xueming Li and Edward H Egelman and Marek Basler and Yifan Cheng and David Baker and Frank DiMaio}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Wang_NatMethods_2015.pdf}, doi = {10.1038/nmeth.3287}, issn = {1548-7105}, year = {2015}, date = {2015-02-01}, journal = {Nature methods}, abstract = {We present a de novo model-building approach that combines predicted backbone conformations with side-chain fit to density to accurately assign sequence into density maps. This method yielded accurate models for six of nine experimental maps at 3.3- to 4.8-r A resolution and produced a nearly complete model for an unsolved map containing a 660-residue heterodimeric protein. This method should enable rapid and reliable protein structure determination from near-atomic-resolution cryo-electron microscopy (cryo-EM) maps.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We present a de novo model-building approach that combines predicted backbone conformations with side-chain fit to density to accurately assign sequence into density maps. This method yielded accurate models for six of nine experimental maps at 3.3- to 4.8-r A resolution and produced a nearly complete model for an unsolved map containing a 660-residue heterodimeric protein. This method should enable rapid and reliable protein structure determination from near-atomic-resolution cryo-electron microscopy (cryo-EM) maps. |
Rossi, Paolo; Shi, Lei; Liu, Gaohua; Barbieri, Christopher M; Lee, Hsiau-Wei; Grant, Thomas D; Luft, Joseph R; Xiao, Rong; Acton, Thomas B; Snell, Edward H; Montelione, Gaetano T; Baker, David; Lange, Oliver F; Sgourakis, Nikolaos G A hybrid NMR/SAXS-based approach for discriminating oligomeric protein interfaces using Rosetta Journal Article Proteins, 83 , pp. 309-17, 2015, ISSN: 1097-0134. @article{611, title = {A hybrid NMR/SAXS-based approach for discriminating oligomeric protein interfaces using Rosetta}, author = { Paolo Rossi and Lei Shi and Gaohua Liu and Christopher M Barbieri and Hsiau-Wei Lee and Thomas D Grant and Joseph R Luft and Rong Xiao and Thomas B Acton and Edward H Snell and Gaetano T Montelione and David Baker and Oliver F Lange and Nikolaos G Sgourakis}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/ahybridnmrsaxsbased_Baker2015.pdf}, doi = {10.1002/prot.24719}, issn = {1097-0134}, year = {2015}, date = {2015-02-01}, journal = {Proteins}, volume = {83}, pages = {309-17}, abstract = {Oligomeric proteins are important targets for structure determination in solution. While in most cases the fold of individual subunits can be determined experimentally, or predicted by homology-based methods, protein-protein interfaces are challenging to determine de novo using conventional NMR structure determination protocols. Here we focus on a member of the bet-V1 superfamily, Aha1 from Colwellia psychrerythraea. This family displays a broad range of crystallographic interfaces none of which can be reconciled with the NMR and SAXS data collected for Aha1. Unlike conventional methods relying on a dense network of experimental restraints, the sparse data are used to limit conformational search during optimization of a physically realistic energy function. This work highlights a new approach for studying minor conformational changes due to structural plasticity within a single dimeric interface in solution.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Oligomeric proteins are important targets for structure determination in solution. While in most cases the fold of individual subunits can be determined experimentally, or predicted by homology-based methods, protein-protein interfaces are challenging to determine de novo using conventional NMR structure determination protocols. Here we focus on a member of the bet-V1 superfamily, Aha1 from Colwellia psychrerythraea. This family displays a broad range of crystallographic interfaces none of which can be reconciled with the NMR and SAXS data collected for Aha1. Unlike conventional methods relying on a dense network of experimental restraints, the sparse data are used to limit conformational search during optimization of a physically realistic energy function. This work highlights a new approach for studying minor conformational changes due to structural plasticity within a single dimeric interface in solution. |
Pearson, Aaron D; Mills, Jeremy H; Song, Yifan; Nasertorabi, Fariborz; Han, Gye Won; Baker, David; Stevens, Raymond C; Schultz, Peter G Transition states. Trapping a transition state in a computationally designed protein bottle. Journal Article Science (New York, N.Y.), 347 , pp. 863-7, 2015, ISSN: 1095-9203. @article{561, title = {Transition states. Trapping a transition state in a computationally designed protein bottle.}, author = { Aaron D Pearson and Jeremy H Mills and Yifan Song and Fariborz Nasertorabi and Gye Won Han and David Baker and Raymond C Stevens and Peter G Schultz}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Mills_Science_2015A.pdf}, doi = {10.1126/science.aaa2424}, issn = {1095-9203}, year = {2015}, date = {2015-02-01}, journal = {Science (New York, N.Y.)}, volume = {347}, pages = {863-7}, abstract = {The fleeting lifetimes of the transition states (TSs) of chemical reactions make determination of their three-dimensional structures by diffraction methods a challenge. Here, we used packing interactions within the core of a protein to stabilize the planar TS conformation for rotation around the central carbon-carbon bond of biphenyl so that it could be directly observed by x-ray crystallography. The computational protein design software Rosetta was used to design a pocket within threonyl-transfer RNA synthetase from the thermophile Pyrococcus abyssi that forms complementary van der Waals interactions with a planar biphenyl. This latter moiety was introduced biosynthetically as the side chain of the noncanonical amino acid p-biphenylalanine. Through iterative rounds of computational design and structural analysis, we identified a protein in which the side chain of p-biphenylalanine is trapped in the energetically disfavored, coplanar conformation of the TS of the bond rotation reaction.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The fleeting lifetimes of the transition states (TSs) of chemical reactions make determination of their three-dimensional structures by diffraction methods a challenge. Here, we used packing interactions within the core of a protein to stabilize the planar TS conformation for rotation around the central carbon-carbon bond of biphenyl so that it could be directly observed by x-ray crystallography. The computational protein design software Rosetta was used to design a pocket within threonyl-transfer RNA synthetase from the thermophile Pyrococcus abyssi that forms complementary van der Waals interactions with a planar biphenyl. This latter moiety was introduced biosynthetically as the side chain of the noncanonical amino acid p-biphenylalanine. Through iterative rounds of computational design and structural analysis, we identified a protein in which the side chain of p-biphenylalanine is trapped in the energetically disfavored, coplanar conformation of the TS of the bond rotation reaction. |
Egelman, E H; Xu, C; DiMaio, F; Magnotti, E; Modlin, C; Yu, X; Wright, E; Baker, D; Conticello, V P Structural plasticity of helical nanotubes based on coiled-coil assemblies. Journal Article Structure (London, England : 1993), 23 , pp. 280-9, 2015, ISSN: 1878-4186. @article{609, title = {Structural plasticity of helical nanotubes based on coiled-coil assemblies.}, author = { E H Egelman and C. Xu and F. DiMaio and E Magnotti and C Modlin and X Yu and E Wright and D Baker and V P Conticello}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/structuralplasticity_Baker2015.pdf}, doi = {10.1016/j.str.2014.12.008}, issn = {1878-4186}, year = {2015}, date = {2015-02-01}, journal = {Structure (London, England : 1993)}, volume = {23}, pages = {280-9}, abstract = {Numerous instances can be seen in evolution in which protein quaternary structures have diverged while the sequences of the building blocks have remained fairly conserved. However, the path through which such divergence has taken place is usually not known. We have designed two synthetic 29-residue α-helical peptides, based on the coiled-coil structural motif, that spontaneously self-assemble into helical nanotubes in vitro. Using electron cryomicroscopy with a newly available direct electron detection capability, we can achieve near-atomic resolution of these thin structures. We show how conservative changes of only one or two amino acids result in dramatic changes in quaternary structure, in which the assemblies can be switched between two very different forms. This system provides a framework for understanding how small sequence changes in evolution can translate into very large changes in supramolecular structure, a phenomenon that may have significant implications for the de novo design of synthetic peptide assemblies.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Numerous instances can be seen in evolution in which protein quaternary structures have diverged while the sequences of the building blocks have remained fairly conserved. However, the path through which such divergence has taken place is usually not known. We have designed two synthetic 29-residue α-helical peptides, based on the coiled-coil structural motif, that spontaneously self-assemble into helical nanotubes in vitro. Using electron cryomicroscopy with a newly available direct electron detection capability, we can achieve near-atomic resolution of these thin structures. We show how conservative changes of only one or two amino acids result in dramatic changes in quaternary structure, in which the assemblies can be switched between two very different forms. This system provides a framework for understanding how small sequence changes in evolution can translate into very large changes in supramolecular structure, a phenomenon that may have significant implications for the de novo design of synthetic peptide assemblies. |
Morag, Omry; Sgourakis, Nikolaos G; Baker, David; Goldbourt, Amir The NMR-Rosetta capsid model of M13 bacteriophage reveals a quadrupled hydrophobic packing epitope Journal Article Proceedings of the National Academy of Sciences of the United States of America, 112 , pp. 971-6, 2015, ISSN: 1091-6490. @article{607, title = {The NMR-Rosetta capsid model of M13 bacteriophage reveals a quadrupled hydrophobic packing epitope}, author = { Omry Morag and Nikolaos G Sgourakis and David Baker and Amir Goldbourt}, url = {http://www.bakerlab.org/wp-content/uploads/2015/12/thenmrrosettacapsid_Baker2015.pdf}, doi = {10.1073/pnas.1415393112}, issn = {1091-6490}, year = {2015}, date = {2015-01-01}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {112}, pages = {971-6}, abstract = {Filamentous phage are elongated semiflexible ssDNA viruses that infect bacteria. The M13 phage, belonging to the family inoviridae, has a length of ~1 μm and a diameter of ~7 nm. Here we present a structural model for the capsid of intact M13 bacteriophage using Rosetta model building guided by structure restraints obtained from magic-angle spinning solid-state NMR experimental data. The C5 subunit symmetry observed in fiber diffraction studies was enforced during model building. The structure consists of stacked pentamers with largely alpha helical subunits containing an N-terminal type II β-turn; there is a rise of 16.6-16.7 r A and a tilt of 36.1-36.6textdegree between consecutive pentamers. The packing of the subunits is stabilized by a repeating hydrophobic stacking pocket; each subunit participates in four pockets by contributing different hydrophobic residues, which are spread along the subunit sequence. Our study provides, to our knowledge, the first magic-angle spinning NMR structure of an intact filamentous virus capsid and further demonstrates the strength of this technique as a method of choice to study noncrystalline, high-molecular-weight molecular assemblies.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Filamentous phage are elongated semiflexible ssDNA viruses that infect bacteria. The M13 phage, belonging to the family inoviridae, has a length of ~1 μm and a diameter of ~7 nm. Here we present a structural model for the capsid of intact M13 bacteriophage using Rosetta model building guided by structure restraints obtained from magic-angle spinning solid-state NMR experimental data. The C5 subunit symmetry observed in fiber diffraction studies was enforced during model building. The structure consists of stacked pentamers with largely alpha helical subunits containing an N-terminal type II β-turn; there is a rise of 16.6-16.7 r A and a tilt of 36.1-36.6textdegree between consecutive pentamers. The packing of the subunits is stabilized by a repeating hydrophobic stacking pocket; each subunit participates in four pockets by contributing different hydrophobic residues, which are spread along the subunit sequence. Our study provides, to our knowledge, the first magic-angle spinning NMR structure of an intact filamentous virus capsid and further demonstrates the strength of this technique as a method of choice to study noncrystalline, high-molecular-weight molecular assemblies. |
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. |