Computational remodeling of an enzyme conformational landscape for altered substrate selectivity

St-Jacques, Antony D.; Rodriguez, Joshua M.; Eason, Matthew G.; Foster, Scott M.; Khan, Safwat T.; Damry, Adam M.; Goto, Natalie K.; Thompson, Michael C.; Chica, Roberto A.

doi:10.1038/s41467-023-41762-0

Computational remodeling of an enzyme conformational landscape for altered substrate selectivity

Journal Article · Thu Sep 28 00:00:00 EDT 2023 · Nature Communications

DOI:https://doi.org/10.1038/s41467-023-41762-0· OSTI ID:2422656

^[1]; Rodriguez, Joshua M. ^[2]; Eason, Matthew G. ^[1]; Foster, Scott M. ^[1]; Khan, Safwat T. ^[1]; Damry, Adam M. ^[1]; Goto, Natalie K. ^[1]; ^[2]; ^[1]

Univ. of Ottawa, ON (Canada)
Univ. of California, Merced, CA (United States)

Structural plasticity of enzymes dictates their function. Yet, our ability to rationally remodel enzyme conformational landscapes to tailor catalytic properties remains limited. Here, we report a computational procedure for tuning conformational landscapes that is based on multistate design of hinge-mediated domain motions. Using this method, we redesign the conformational landscape of a natural aminotransferase to preferentially stabilize a less populated but reactive conformation and thereby increase catalytic efficiency with a non-native substrate, resulting in altered substrate selectivity. Steady-state kinetics of designed variants reveals activity increases with the non-native substrate of approximately 100-fold and selectivity switches of up to 1900-fold. Structural analyses by room-temperature X-ray crystallography and multitemperature nuclear magnetic resonance spectroscopy confirm that conformational equilibria favor the target conformation. Our computational approach opens the door to targeted alterations of conformational states and equilibria, which should facilitate the design of biocatalysts with customized activity and selectivity.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States). Advanced Light Source (ALS)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institutes of Health (NIH); Natural Sciences and Engineering Research Council of Canada (NSERC); Ontario Ministry of Economic Development & Innovation; Canada Foundation for Innovation (CFI)

Grant/Contract Number:: AC02-05CH11231; AC02-76SF00515

OSTI ID:: 2422656

Journal Information:: Nature Communications, Journal Name: Nature Communications Journal Issue: 1 Vol. 14; ISSN 2041-1723

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

References (61)

LMFIT: Non-Linear Least-Square Minimization and Curve-Fitting for Python Newville, Matthew; Stensitzki, Till; Allen, Daniel B. Zenodo https://doi.org/10.5281/zenodo.11813	software	September 2014
How well does a restrained electrostatic potential (RESP) model perform in calculating conformational energies of organic and biological molecules? Wang, Junmei; Cieplak, Piotr; Kollman, Peter A. Journal of Computational Chemistry, Vol. 21, Issue 12 https://doi.org/10.1002/1096-987X(200009)21:12<1049::AID-JCC3>3.0.CO;2-F	journal	January 2000
An efficient algorithm for multistate protein design based on FASTER Allen, Benjamin D.; Mayo, Stephen L. Journal of Computational Chemistry, Vol. 31, Issue 5 https://doi.org/10.1002/jcc.21375	journal	July 2009
Multistate approaches in computational protein design: Multistate Computational Protein Design Davey, James A.; Chica, Roberto A. Protein Science, Vol. 21, Issue 9 https://doi.org/10.1002/pro.2128	journal	August 2012
Redesign of the substrate specificity of escherichia coli aspartate aminotransferase to that of escherichia coli tyrosine aminotransferase by homology modeling and site-directed mutagenesis Onuffer, James J.; Kirsch, Jack F. Protein Science, Vol. 4, Issue 9 https://doi.org/10.1002/pro.5560040910	journal	September 1995
Improving the accuracy of protein stability predictions with multistate design using a variety of backbone ensembles: Improving Multistate Design Predictions Davey, James A.; Chica, Roberto A. Proteins: Structure, Function, and Bioinformatics, Vol. 82, Issue 5 https://doi.org/10.1002/prot.24457	journal	November 2013
Crystal Structures of Escherichia coli Aspartate Aminotransferase in Two Conformations Jäger, Joachim; Moser, Markus; Sauder, Ursula Journal of Molecular Biology, Vol. 239, Issue 2 https://doi.org/10.1006/jmbi.1994.1368	journal	June 1994
A survey of truncated-Newton methods Nash, Stephen G. Journal of Computational and Applied Mathematics, Vol. 124, Issue 1-2 https://doi.org/10.1016/S0377-0427(00)00426-X	journal	December 2000
Rotamer Libraries in the 21st Century Dunbrack, Roland L. Current Opinion in Structural Biology, Vol. 12, Issue 4 https://doi.org/10.1016/S0959-440X(02)00344-5	journal	August 2002
Improvements in the analysis of domain motions in proteins from conformational change: DynDom version 1.50 Hayward, Steven; Lee, Richard A. Journal of Molecular Graphics and Modelling, Vol. 21, Issue 3 https://doi.org/10.1016/S1093-3263(02)00140-7	journal	December 2002
Ancestral Protein Reconstruction Yields Insights into Adaptive Evolution of Binding Specificity in Solute-Binding Proteins Clifton, Ben E.; Jackson, Colin J. Cell Chemical Biology, Vol. 23, Issue 2 https://doi.org/10.1016/j.chembiol.2015.12.010	journal	February 2016
Janus: Prediction and Ranking of Mutations Required for Functional Interconversion of Enzymes Addington, Trevor A.; Mertz, Robert W.; Siegel, Justin B. Journal of Molecular Biology, Vol. 425, Issue 8 https://doi.org/10.1016/j.jmb.2013.01.034	journal	April 2013
Characterizing and Predicting Protein Hinges for Mechanistic Insight Khade, Pranav M.; Kumar, Ambuj; Jernigan, Robert L. Journal of Molecular Biology, Vol. 432, Issue 2 https://doi.org/10.1016/j.jmb.2019.11.018	journal	January 2020
Crystal Cryocooling Distorts Conformational Heterogeneity in a Model Michaelis Complex of DHFR Keedy, Daniel A.; van den Bedem, Henry; Sivak, David A. Structure, Vol. 22, Issue 6 https://doi.org/10.1016/j.str.2014.04.016	journal	June 2014
Measuring Excess Heat Capacities of Deoxyribonucleic Acid (DNA) Folding at the Single-Molecule Level Nicholson, David A.; Jia, Bin; Nesbitt, David J. The Journal of Physical Chemistry B, Vol. 125, Issue 34 https://doi.org/10.1021/acs.jpcb.1c05555	journal	August 2021
Pre-steady-state kinetics of Escherichia coli aspartate aminotransferase catalyzed reactions and thermodynamic aspects of its substrate specificity Kuramitsu, Seiki; Hiromi, Keitaro; Hayashi, Hideyuki Biochemistry, Vol. 29, Issue 23 https://doi.org/10.1021/bi00475a010	journal	June 1990
Binding of C5-Dicarboxylic Substrate to Aspartate Aminotransferase: Implications for the Conformational Change at the Transaldimination Step^, Islam, Mohammad Mainul; Goto, Masaru; Miyahara, Ikuko Biochemistry, Vol. 44, Issue 23 https://doi.org/10.1021/bi050071g	journal	May 2005
Free-Energy Landscape of Enzyme Catalysis ^† Benkovic, Stephen J.; Hammes, Gordon G.; Hammes-Schiffer, Sharon Biochemistry, Vol. 47, Issue 11 https://doi.org/10.1021/bi800049z	journal	March 2008
The Reaction Catalyzed by Escherichia coli Aspartate Aminotransferase Has Multiple Partially Rate-Determining Steps, While That Catalyzed by the Y225F Mutant Is Dominated by Ketimine Hydrolysis ^† Goldberg, Jonathan M.; Kirsch, Jack F. Biochemistry, Vol. 35, Issue 16 https://doi.org/10.1021/bi952138d	journal	January 1996
DREIDING: a generic force field for molecular simulations Mayo, Stephen L.; Olafson, Barry D.; Goddard, William A. The Journal of Physical Chemistry, Vol. 94, Issue 26, p. 8897-8909 https://doi.org/10.1021/j100389a010	journal	December 1990
Bioanalysis for Biocatalysis: Multiplexed Capillary Electrophoresis–Mass Spectrometry Assay for Aminotransferase Substrate Discovery and Specificity Profiling Mironov, Gleb G.; St-Jacques, Antony D.; Mungham, Alexander Journal of the American Chemical Society, Vol. 135, Issue 37 https://doi.org/10.1021/ja407486z	journal	September 2013
Enzyme Substrate Specificity Conferred by Distinct Conformational Pathways Rago, Florencia; Saltzberg, Daniel; Allen, Karen N. Journal of the American Chemical Society, Vol. 137, Issue 43 https://doi.org/10.1021/jacs.5b08149	journal	October 2015
Directed Evolution Mimics Allosteric Activation by Stepwise Tuning of the Conformational Ensemble Buller, Andrew R.; van Roye, Paul; Cahn, Jackson K. B. Journal of the American Chemical Society, Vol. 140, Issue 23 https://doi.org/10.1021/jacs.8b03490	journal	April 2018
Deciphering the Allosterically Driven Conformational Ensemble in Tryptophan Synthase Evolution Maria-Solano, Miguel A.; Iglesias-Fernández, Javier; Osuna, Sílvia Journal of the American Chemical Society, Vol. 141, Issue 33 https://doi.org/10.1021/jacs.9b03646	journal	July 2019
Dynamic personalities of proteins Henzler-Wildman, Katherine; Kern, Dorothee Nature, Vol. 450, Issue 7172 https://doi.org/10.1038/nature06522	journal	December 2007
Hidden alternative structures of proline isomerase essential for catalysis Fraser, James S.; Clarkson, Michael W.; Degnan, Sheena C. Nature, Vol. 462, Issue 7273 https://doi.org/10.1038/nature08615	journal	December 2009
The role of protein dynamics in the evolution of new enzyme function Campbell, Eleanor; Kaltenbach, Miriam; Correy, Galen J. Nature Chemical Biology, Vol. 12, Issue 11 https://doi.org/10.1038/nchembio.2175	journal	September 2016
Gene splicing and mutagenesis by PCR-driven overlap extension Heckman, Karin L.; Pease, Larry R. Nature Protocols, Vol. 2, Issue 4 https://doi.org/10.1038/nprot.2007.132	journal	April 2007
Preparation of site-specifically labeled fluorinated proteins for 19F-NMR structural characterization Hammill, Jared T.; Miyake-Stoner, Shigeki; Hazen, Jennifer L. Nature Protocols, Vol. 2, Issue 10 https://doi.org/10.1038/nprot.2007.379	journal	October 2007
Alternating arginine-modulated substrate specificity in an engineered tyrosine aminotransferase Malashkevich, Vladimir N.; Onuffer, James J.; Kirsch, Jack F. Nature Structural Biology, Vol. 2, Issue 7 https://doi.org/10.1038/nsb0795-548	journal	July 1995
Manipulation of ligand binding affinity by exploitation of conformational coupling Marvin, Jonathan S.; Hellinga, Homme W. Nature Structural Biology, Vol. 8, Issue 9, p. 795-798 https://doi.org/10.1038/nsb0901-795	journal	September 2001
The energy landscape of adenylate kinase during catalysis Kerns, S. Jordan; Agafonov, Roman V.; Cho, Young-Jin Nature Structural & Molecular Biology, Vol. 22, Issue 2 https://doi.org/10.1038/nsmb.2941	journal	January 2015
The evolution of multiple active site configurations in a designed enzyme Hong, Nan-Sook; Petrović, Dušan; Lee, Richmond Nature Communications, Vol. 9, Issue 1 https://doi.org/10.1038/s41467-018-06305-y	journal	September 2018
Altered conformational sampling along an evolutionary trajectory changes the catalytic activity of an enzyme Kaczmarski, Joe A.; Mahawaththa, Mithun C.; Feintuch, Akiva Nature Communications, Vol. 11, Issue 1 https://doi.org/10.1038/s41467-020-19695-9	journal	November 2020
Evolution of chalcone isomerase from a noncatalytic ancestor Kaltenbach, Miriam; Burke, Jason R.; Dindo, Mirco Nature Chemical Biology, Vol. 14, Issue 6 https://doi.org/10.1038/s41589-018-0042-3	journal	April 2018
Evolution of cyclohexadienyl dehydratase from an ancestral solute-binding protein Clifton, Ben E.; Kaczmarski, Joe A.; Carr, Paul D. Nature Chemical Biology, Vol. 14, Issue 6 https://doi.org/10.1038/s41589-018-0043-2	journal	April 2018
Role of conformational dynamics in the evolution of novel enzyme function Maria-Solano, Miguel A.; Serrano-Hervás, Eila; Romero-Rivera, Adrian Chemical Communications, Vol. 54, Issue 50 https://doi.org/10.1039/C8CC02426J	journal	January 2018
Experimental library screening demonstrates the successful application of computational protein design to large structural ensembles Allen, B. D.; Nisthal, A.; Mayo, S. L. Proceedings of the National Academy of Sciences, Vol. 107, Issue 46 https://doi.org/10.1073/pnas.1012985107	journal	November 2010
Generation of longer emission wavelength red fluorescent proteins using computationally designed libraries Chica, R. A.; Moore, M. M.; Allen, B. D. Proceedings of the National Academy of Sciences, Vol. 107, Issue 47 https://doi.org/10.1073/pnas.1013910107	journal	November 2010
Iterative approach to computational enzyme design Privett, H. K.; Kiss, G.; Lee, T. M. Proceedings of the National Academy of Sciences, Vol. 109, Issue 10 https://doi.org/10.1073/pnas.1118082108	journal	February 2012
Computational design of closely related proteins that adopt two well-defined but structurally divergent folds Wei, Kathy Y.; Moschidi, Danai; Bick, Matthew J. Proceedings of the National Academy of Sciences, Vol. 117, Issue 13 https://doi.org/10.1073/pnas.1914808117	journal	March 2020
Probing the role of packing specificity in protein design Dahiyat, B. I.; Mayo, S. L. Proceedings of the National Academy of Sciences, Vol. 94, Issue 19 https://doi.org/10.1073/pnas.94.19.10172	journal	September 1997
Refinement and Comparisons of the Crystal Structures of Pig Cytosolic Aspartate Aminotransferase and Its Complex with 2-Methylaspartate Rhee, Sangkee; Silva, Mercedes M.; Hyde, C. Craig Journal of Biological Chemistry, Vol. 272, Issue 28 https://doi.org/10.1074/jbc.272.28.17293	journal	July 1997
Thermodynamic Analysis Reveals a Temperature-dependent Change in the Catalytic Mechanism of Bacillus stearothermophilus Tyrosyl-tRNA Synthetase Sharma, Gyanesh; First, Eric A. Journal of Biological Chemistry, Vol. 284, Issue 7 https://doi.org/10.1074/jbc.M808500200	journal	February 2009
Tyrosine nitration on calmodulin enhances calcium-dependent association and activation of nitric-oxide synthase Porter, Joseph J.; Jang, Hyo Sang; Haque, Mohammad Mahfuzul Journal of Biological Chemistry, Vol. 295, Issue 8 https://doi.org/10.1074/jbc.RA119.010999	journal	February 2020
X-Ray Crystallographic Study of Pyridoxal 5′-Phosphate-Type Aspartate Aminotransferases from Escherichia coli in Open and Closed Form1 Okamoto, Akihiro; Higuchi, Taiichi; Hirotsu, Ken The Journal of Biochemistry, Vol. 116, Issue 1 https://doi.org/10.1093/oxfordjournals.jbchem.a124509	journal	July 1994
X-Ray Crystallographic Study of Pyridoxamine 5'-Phosphate-Type Aspartate Aminotransferases from Escherichia coli in Three Forms1 Miyahara, Ikuko; Hirotsu, Ken; Hayashi, Hideyuki The Journal of Biochemistry, Vol. 116, Issue 5 https://doi.org/10.1093/oxfordjournals.jbchem.a124620	journal	November 1994
Phaser crystallographic software McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D. Journal of Applied Crystallography, Vol. 40, Issue 4 https://doi.org/10.1107/S0021889807021206	journal	July 2007
xia2 : an expert system for macromolecular crystallography data reduction Winter, G. Journal of Applied Crystallography, Vol. 43, Issue 1 https://doi.org/10.1107/S0021889809045701	journal	December 2009
electronic Ligand Builder and Optimization Workbench ( eLBOW ): a tool for ligand coordinate and restraint generation Moriarty, Nigel W.; Grosse-Kunstleve, Ralf W.; Adams, Paul D. Acta Crystallographica Section D Biological Crystallography, Vol. 65, Issue 10 https://doi.org/10.1107/S0907444909029436	journal	September 2009
PHENIX: a comprehensive Python-based system for macromolecular structure solution Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221 https://doi.org/10.1107/S0907444909052925	journal	January 2010
Towards automated crystallographic structure refinement with phenix.refine Afonine, Pavel V.; Grosse-Kunstleve, Ralf W.; Echols, Nathaniel Acta Crystallographica Section D Biological Crystallography, Vol. 68, Issue 4 https://doi.org/10.1107/S0907444912001308	journal	March 2012
DIALS : implementation and evaluation of a new integration package Winter, Graeme; Waterman, David G.; Parkhurst, James M. Acta Crystallographica Section D Structural Biology, Vol. 74, Issue 2 https://doi.org/10.1107/S2059798317017235	journal	February 2018
Scaling diffraction data in the DIALS software package: algorithms and new approaches for multi-crystal scaling Beilsten-Edmands, James; Winter, Graeme; Gildea, Richard Acta Crystallographica Section D Structural Biology, Vol. 76, Issue 4 https://doi.org/10.1107/S2059798320003198	journal	March 2020
Propensities, probabilities, and the Boltzmann hypothesis Shortle, David Protein Science, Vol. 12, Issue 6 https://doi.org/10.1110/ps.0306903	journal	June 2003
The Protein Data Bank. A Computer-Based Archival File for Macromolecular Structures Bernstein, Frances C.; Koetzle, Thomas F.; Williams, Graheme J. B. European Journal of Biochemistry, Vol. 80, Issue 2 https://doi.org/10.1111/j.1432-1033.1977.tb11885.x	journal	November 1977
The Dynamic Energy Landscape of Dihydrofolate Reductase Catalysis Boehr, D. D.; McElheny, D.; Dyson, H. J. Science, Vol. 313, Issue 5793, p. 1638-1642 https://doi.org/10.1126/science.1130258	journal	September 2006
Protein Dynamism and Evolvability Tokuriki, N.; Tawfik, D. S. Science, Vol. 324, Issue 5924 https://doi.org/10.1126/science.1169375	journal	April 2009
How directed evolution reshapes the energy landscape in an enzyme to boost catalysis Otten, Renee; Pádua, Ricardo A. P.; Bunzel, H. Adrian Science, Vol. 370, Issue 6523 https://doi.org/10.1126/science.abd3623	journal	December 2020
Pyridoxal Phosphate Enzymes: Mechanistic, Structural, and Evolutionary Considerations Eliot, Andrew C.; Kirsch, Jack F. Annual Review of Biochemistry, Vol. 73, Issue 1 https://doi.org/10.1146/annurev.biochem.73.011303.074021	journal	June 2004
Linearization of the Bradford Protein Assay Ernst, Orna; Zor, Tsaffrir Journal of Visualized Experiments, Issue 38 https://doi.org/10.3791/1918-v	journal	April 2010

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