Mix-and-inject XFEL crystallography reveals gated conformational dynamics during enzyme catalysis

Dasgupta, Medhanjali; Budday, Dominik; de Oliveira, Saulo H.  P.; Madzelan, Peter; Marchany-Rivera, Darya; Seravalli, Javier; Hayes, Brandon; Sierra, Raymond G.; Boutet, Sébastien; Hunter, Mark S.; Alonso-Mori, Roberto; Batyuk, Alexander; Wierman, Jennifer; Lyubimov, Artem; Brewster, Aaron S.; Sauter, Nicholas K.; Applegate, Gregory A.; Tiwari, Virendra K.; Berkowitz, David B.; Thompson, Michael C.; Cohen, Aina E.; Fraser, James S.; Wall, Michael E.; van den Bedem, Henry; Wilson, Mark A.

doi:10.1073/pnas.1901864116

Mix-and-inject XFEL crystallography reveals gated conformational dynamics during enzyme catalysis

Journal Article · Tue Dec 03 23:00:00 EST 2019 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.1901864116· OSTI ID:1604575

Dasgupta, Medhanjali ^[1]; Budday, Dominik ^[2]; de Oliveira, Saulo H. P. ^[3]; Madzelan, Peter ^[1]; Marchany-Rivera, Darya ^[4]; Seravalli, Javier ^[1]; Hayes, Brandon ^[5]; Sierra, Raymond G. ^[6]; Boutet, Sébastien ^[5]; Hunter, Mark S. ^[5]; Alonso-Mori, Roberto ^[5]; ^[5]; Wierman, Jennifer ^[7]; Lyubimov, Artem ^[7]; Brewster, Aaron S. ^[8]; Sauter, Nicholas K. ^[8]; Applegate, Gregory A. ^[1]; Tiwari, Virendra K. ^[1]; Berkowitz, David B. ^[1]; Thompson, Michael C. ^[9] more »

Univ. of Nebraska, Lincoln, NE (United States)
Friedrich-Alexander Univ., Erlangen (Germany)
Stanford Univ., CA (United States); SLAC National Accelerator Lab., Menlo Park, CA (United States)
Univ. of Puerto Rico, Mayaguez (Puerto Rico)
SLAC National Accelerator Lab., Menlo Park, CA (United States). Linac Coherent Light Source (LCLS)
SLAC National Accelerator Lab., Menlo Park, CA (United States). Linac Coherent Light Source (LCLS) and Photon Ultrafast Laser Science and Engineering Inst. (PULSE)
SLAC National Accelerator Lab., Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL)
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Univ. of California, San Francisco, CA (United States)
Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
SLAC National Accelerator Lab., Menlo Park, CA (United States); Univ. of California, San Francisco, CA (United States)

How changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question. Here, we use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL), ambient-temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent catalysis modulates isocyanide hydratase (ICH) conformational dynamics throughout its catalytic cycle. We visualize this previously hypothetical reaction mechanism, directly observing formation of a thioimidate covalent intermediate in ICH microcrystals during catalysis. We report, ICH exhibits a concerted helical displacement upon active-site cysteine modification that is gated by changes in hydrogen bond strength between the cysteine thiolate and the backbone amide of the highly strained Ile152 residue. These catalysis-activated motions permit water entry into the ICH active site for intermediate hydrolysis. Mutations at a Gly residue (Gly150) that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady-state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. These results demonstrate that MISC can capture otherwise elusive aspects of enzyme mechanism and dynamics in microcrystalline samples, resolving long-standing questions about the connection between nonequilibrium protein motions and enzyme catalysis.

View Accepted Manuscript (DOE)

Research Organization:: SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL) and Linac Coherent Light Source (LCLS); Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences & Biosciences Division; USDOE Laboratory Directed Research and Development (LDRD) Program; National Institutes of Health (NIH); National Science Foundation (NSF); David and Lucile Packard Foundation; University of California (UC) Office of the President Laboratory Fees Research Program; USDOE National Nuclear Security Administration (NNSA); Deutsche Forschungsgemeinschaft (DFG); Nebraska Tobacco Settlement Biomedical Research Development Fund; USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institute of General Medical Sciences (NIGMS); Planck Institute for Medical Research

Grant/Contract Number:: AC02-76SF00515; AC02-06CH11357

OSTI ID:: 1604575

Alternate ID(s):: OSTI ID: 1633800
OSTI ID: 1659760

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 51 Vol. 116; ISSN 0027-8424

Publisher:: National Academy of SciencesCopyright Statement

Country of Publication:: United States

Language:: English

References (55)

‘Negative’ and ‘positive catalysis’: complementary principles that shape the catalytic landscape of enzymes Vögeli, Bastian; Erb, Tobias J. Current Opinion in Chemical Biology, Vol. 47 https://doi.org/10.1016/j.cbpa.2018.09.013	journal	December 2018
Perspective: Defining and quantifying the role of dynamics in enzyme catalysis Warshel, Arieh; Bora, Ram Prasad The Journal of Chemical Physics, Vol. 144, Issue 18 https://doi.org/10.1063/1.4947037	journal	May 2016
Direct observation of ultrafast large-scale dynamics of an enzyme under turnover conditions Aviram, Haim Yuval; Pirchi, Menahem; Mazal, Hisham Proceedings of the National Academy of Sciences, Vol. 115, Issue 13 https://doi.org/10.1073/pnas.1720448115	journal	March 2018
The Amber biomolecular simulation programs Case, David A.; Cheatham, Thomas E.; Darden, Tom Journal of Computational Chemistry, Vol. 26, Issue 16 https://doi.org/10.1002/jcc.20290	journal	January 2005
The 1.0 Å crystal structure of Ca2+-bound calmodulin: an analysis of disorder and implications for functionally relevant plasticity Wilson, Mark A.; Brunger, Axel T. Journal of Molecular Biology, Vol. 301, Issue 5 https://doi.org/10.1006/jmbi.2000.4029	journal	September 2000
[20] Processing of X-ray diffraction data collected in oscillation mode Otwinowski, Zbyszek; Minor, Wladek Macromolecular Crystallography Part A, 307-326 https://doi.org/10.1016/S0076-6879(97)76066-X	book	January 1997
Serial femtosecond crystallography: A revolution in structural biology Martin-Garcia, Jose M.; Conrad, Chelsie E.; Coe, Jesse Archives of Biochemistry and Biophysics, Vol. 602 https://doi.org/10.1016/j.abb.2016.03.036	journal	July 2016
Expanding the functional diversity of proteins through cysteine oxidation Reddie, Khalilah G.; Carroll, Kate S. Current Opinion in Chemical Biology, Vol. 12, Issue 6 https://doi.org/10.1016/j.cbpa.2008.07.028	journal	December 2008
Automatic atom type and bond type perception in molecular mechanical calculations Wang, Junmei; Wang, Wei; Kollman, Peter A. Journal of Molecular Graphics and Modelling, Vol. 25, Issue 2 https://doi.org/10.1016/j.jmgm.2005.12.005	journal	October 2006
Bringing diffuse X-ray scattering into focus Wall, Michael E.; Wolff, Alexander M.; Fraser, James S. Current Opinion in Structural Biology, Vol. 50 https://doi.org/10.1016/j.sbi.2018.01.009	journal	June 2018
Kinematic Flexibility Analysis: Hydrogen Bonding Patterns Impart a Spatial Hierarchy of Protein Motion Budday, Dominik; Leyendecker, Sigrid; van den Bedem, Henry Journal of Chemical Information and Modeling, Vol. 58, Issue 10 https://doi.org/10.1021/acs.jcim.8b00267	journal	September 2018
qFit-ligand Reveals Widespread Conformational Heterogeneity of Drug-Like Molecules in X-Ray Electron Density Maps van Zundert, Gydo C. P.; Hudson, Brandi M.; de Oliveira, Saulo H. P. Journal of Medicinal Chemistry, Vol. 61, Issue 24 https://doi.org/10.1021/acs.jmedchem.8b01292	journal	November 2018
Structural heterogeneity in protein crystals Smith, Janet L.; Hendrickson, Wayne A.; Honzatko, Richard B. Biochemistry, Vol. 25, Issue 18 https://doi.org/10.1021/bi00366a008	journal	September 1986
Multiple Conformational Changes in Enzyme Catalysis ^† Hammes, Gordon G. Biochemistry, Vol. 41, Issue 26 https://doi.org/10.1021/bi0260839	journal	July 2002
Diverse Functional Roles of Reactive Cysteines Pace, Nicholas J.; Weerapana, Eranthie ACS Chemical Biology, Vol. 8, Issue 2 https://doi.org/10.1021/cb3005269	journal	November 2012
Conformational Gating of Electron Transfer from the Nitrogenase Fe Protein to MoFe Protein Danyal, Karamatullah; Mayweather, Diana; Dean, Dennis R. Journal of the American Chemical Society, Vol. 132, Issue 20 https://doi.org/10.1021/ja101737f	journal	May 2010
Intrinsic motions along an enzymatic reaction trajectory Henzler-Wildman, Katherine A.; Thai, Vu; Lei, Ming Nature, Vol. 450, Issue 7171 https://doi.org/10.1038/nature06410	journal	November 2007
Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography Stagno, J. R.; Liu, Y.; Bhandari, Y. R. Nature, Vol. 541, Issue 7636 https://doi.org/10.1038/nature20599	journal	November 2016
Automated identification of functional dynamic contact networks from X-ray crystallography van den Bedem, Henry; Bhabha, Gira; Yang, Kun Nature Methods, Vol. 10, Issue 9 https://doi.org/10.1038/nmeth.2592	journal	August 2013
Integrative, dynamic structural biology at atomic resolution—it's about time van den Bedem, Henry; Fraser, James S. Nature Methods, Vol. 12, Issue 4 https://doi.org/10.1038/nmeth.3324	journal	March 2015
Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II Sierra, Raymond G.; Gati, Cornelius; Laksmono, Hartawan Nature Methods, Vol. 13, Issue 1 https://doi.org/10.1038/nmeth.3667	journal	November 2015
Is one solution good enough? Furnham, Nicholas; Blundell, Tom L.; DePristo, Mark A. Nature Structural & Molecular Biology, Vol. 13, Issue 3 https://doi.org/10.1038/nsmb0306-184	journal	March 2006
Capturing an initial intermediate during the P450nor enzymatic reaction using time-resolved XFEL crystallography and caged-substrate Tosha, Takehiko; Nomura, Takashi; Nishida, Takuma Nature Communications, Vol. 8, Article No. 1585 https://doi.org/10.1038/s41467-017-01702-1	journal	November 2017
Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation Tillu, Vikas A.; Rae, James; Gao, Ya Nature Communications, Vol. 12, Issue 1 https://doi.org/10.1038/s41467-021-21035-4	journal	February 2021
Structure-based design of targeted covalent inhibitors Lonsdale, Richard; Ward, Richard A. Chemical Society Reviews, Vol. 47, Issue 11 https://doi.org/10.1039/C7CS00220C	journal	January 2018
Structural enzymology using X-ray free electron lasers Kupitz, Christopher; Olmos, Jose L.; Holl, Mark Structural Dynamics, Vol. 4, Issue 4 https://doi.org/10.1063/1.4972069	journal	December 2016
Structural and dynamical description of the enzymatic reaction of a phosphohexomutase Stiers, Kyle M.; Graham, Abigail C.; Zhu, Jian-She Structural Dynamics, Vol. 6, Issue 2 https://doi.org/10.1063/1.5092803	journal	March 2019
Efficient coupling of catalysis and dynamics in the E1 component of Escherichia coli pyruvate dehydrogenase multienzyme complex Kale, S.; Ulas, G.; Song, J. Proceedings of the National Academy of Sciences, Vol. 105, Issue 4 https://doi.org/10.1073/pnas.0709328105	journal	January 2008
Accessing protein conformational ensembles using room-temperature X-ray crystallography Fraser, J. S.; van den Bedem, H.; Samelson, A. J. Proceedings of the National Academy of Sciences, Vol. 108, Issue 39 https://doi.org/10.1073/pnas.1111325108	journal	September 2011
Discovery of a Novel Enzyme, Isonitrile Hydratase, Involved in Nitrogen-Carbon Triple Bond Cleavage Goda, Masahiko; Hashimoto, Yoshiteru; Shimizu, Sakayu Journal of Biological Chemistry, Vol. 276, Issue 26 https://doi.org/10.1074/jbc.M007856200	journal	April 2001
Evolution of New Enzymatic Function by Structural Modulation of Cysteine Reactivity in Pseudomonas fluorescens Isocyanide Hydratase Lakshminarasimhan, Mahadevan; Madzelan, Peter; Nan, Ruth Journal of Biological Chemistry, Vol. 285, Issue 38 https://doi.org/10.1074/jbc.M110.147934	journal	July 2010
MolProbity: all-atom contacts and structure validation for proteins and nucleic acids Davis, I. W.; Leaver-Fay, A.; Chen, V. B. Nucleic Acids Research, Vol. 35, Issue Web Server https://doi.org/10.1093/nar/gkm216	journal	May 2007
Diffraction before destruction Chapman, Henry N.; Caleman, Carl; Timneanu, Nicusor Philosophical Transactions of the Royal Society B: Biological Sciences, Vol. 369, Issue 1647 https://doi.org/10.1098/rstb.2013.0313	journal	July 2014
Coot model-building tools for molecular graphics Emsley, Paul; Cowtan, Kevin Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12, p. 2126-2132 https://doi.org/10.1107/S0907444904019158	journal	November 2004
Modeling discrete heterogeneity in X-ray diffraction data by fitting multi-conformers van den Bedem, Henry; Dhanik, Ankur; Latombe, Jean-Claude Acta Crystallographica Section D Biological Crystallography, Vol. 65, Issue 10 https://doi.org/10.1107/S0907444909030613	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
XFELs for structure and dynamics in biology Spence, J. C. H. IUCrJ, Vol. 4, Issue 4 https://doi.org/10.1107/S2052252517005760	journal	May 2017
Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography Stagno, J. R.; Liu, Y.; Bhandari, Y. R. Acta Crystallographica Section A Foundations and Advances, Vol. 73, Issue a1 https://doi.org/10.1107/s0108767317099081	journal	May 2017
Integration, scaling, space-group assignment and post-refinement Kabsch, Wolfgang Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 133-144 https://doi.org/10.1107/s0907444909047374	journal	January 2010
New Python-based methods for data processing Sauter, Nicholas K.; Hattne, Johan; Grosse-Kunstleve, Ralf W. Acta Crystallographica Section D Biological Crystallography, Vol. 69, Issue 7 https://doi.org/10.1107/s0907444913000863	journal	June 2013
Native proteins trap high-energy transit conformations Brereton, Andrew E.; Karplus, P. Andrew Science Advances, Vol. 1, Issue 9 https://doi.org/10.1126/sciadv.1501188	journal	October 2015
The role of dimer asymmetry and protomer dynamics in enzyme catalysis Kim, Tae Hun; Mehrabi, Pedram; Ren, Zhong Science, Vol. 355, Issue 6322 https://doi.org/10.1126/science.aag2355	journal	January 2017
Time-resolved crystallography reveals allosteric communication aligned with molecular breathing Mehrabi, Pedram; Schulz, Eike C.; Dsouza, Raison Science, Vol. 365, Issue 6458 https://doi.org/10.1126/science.aaw9904	journal	September 2019
Enzyme intermediates captured “on the fly” by mix-and-inject serial crystallography Olmos, Jose L.; Pandey, Suraj; Martin-Garcia, Jose M. BMC Biology, Vol. 16, Issue 1 https://doi.org/10.1186/s12915-018-0524-5	journal	May 2018
MolProbity: all-atom contacts and structure validation for proteins and nucleic acids B., Chen, Vincent; C., Richardson, David; S., Richardson, Jane The University of North Carolina at Chapel Hill University Libraries https://doi.org/10.17615/m2an-p252	text	January 2007
PHENIX: a comprehensive Python-based system for macromolecular structure solution. Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor Apollo - University of Cambridge Repository https://doi.org/10.17863/cam.45787	text	January 2010
Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography Stagno, J. R.; Liu, Y.; Bhandari, Y. R. Deutsches Elektronen-Synchrotron, DESY, Hamburg https://doi.org/10.3204/pubdb-2016-05392	text	January 2016
Structural enzymology using X-ray free electron lasers Kupitz, Christopher; Olmos, Jose L.; Holl, Mark Deutsches Elektronen-Synchrotron, DESY, Hamburg https://doi.org/10.3204/pubdb-2016-06266	text	January 2017
Kinematic Flexibility Analysis: Hydrogen Bonding Patterns Impart a Spatial Hierarchy of Protein Motion Budday, Dominik; Leyendecker, Sigrid; Bedem, Henry van den arXiv https://doi.org/10.48550/arxiv.1802.08683	preprint	January 2018
Qfit-Ligand Reveals Widespread Conformational Heterogeneity Of Drug-Like Molecules In X-Ray Electron Density Maps van Zundert, Gydo C. P.; Hudson, Brandi M.; Keedy, Daniel A. Zenodo https://doi.org/10.5281/zenodo.1256261	dataset	January 2018
Qfit-Ligand Reveals Widespread Conformational Heterogeneity Of Drug-Like Molecules In X-Ray Electron Density Maps van Zundert, Gydo C. P.; Hudson, Brandi M.; Keedy, Daniel A. Zenodo https://doi.org/10.5281/zenodo.1256262	dataset	January 2018
Kinematic Flexibility Analysis: Hydrogen Bonding Patterns Impart A Spatial Hierarchy Of Protein Motion Budday, Dominik; Leyendecker, Sigrid; van den Bedem, Henry Zenodo https://doi.org/10.5281/zenodo.1315003	dataset	January 2018
Kinematic Flexibility Analysis: Hydrogen Bonding Patterns Impart A Spatial Hierarchy Of Protein Motion Budday, Dominik; Leyendecker, Sigrid; van den Bedem, Henry Zenodo https://doi.org/10.5281/zenodo.1315004	dataset	January 2018
Modelling dynamics in protein crystal structures by ensemble refinement Burnley, B. Tom; Afonine, Pavel V.; Adams, Paul D. eLife, Vol. 1 https://doi.org/10.7554/eLife.00311	journal	December 2012
Enabling X-ray free electron laser crystallography for challenging biological systems from a limited number of crystals Uervirojnangkoorn, Monarin; Zeldin, Oliver B.; Lyubimov, Artem Y. eLife, Vol. 4 https://doi.org/10.7554/elife.05421	journal	March 2015

Cited By (1)

Shining light on cysteine modification: connecting protein conformational dynamics to catalysis and regulation van den Bedem, Henry; Wilson, Mark A. Journal of Synchrotron Radiation, Vol. 26, Issue 4 https://doi.org/10.1107/S160057751900568X	journal	June 2019

Similar Records

Changes in an enzyme ensemble during catalysis observed by high-resolution XFEL crystallography

Journal Article · Wed Mar 27 00:00:00 EDT 2024 · Science Advances · OSTI ID:2396810

Related Subjects

36 MATERIALS SCIENCE
X-ray crystallography
XFEL
cysteine modification
dynamics
enzyme conformational
radiation damage

Mix-and-inject XFEL crystallography reveals gated conformational dynamics during enzyme catalysis

Citation Formats

References (55)

Cited By (1)

Similar Records

Related Subjects