skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

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

Abstract

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.

Authors:
 [1];  [2];  [3];  [1];  [4];  [1];  [5];  [6];  [5];  [5];  [5]; ORCiD logo [5];  [7];  [7];  [8];  [8];  [1];  [1];  [1];  [9] more »;  [7];  [9]; ORCiD logo [10]; ORCiD logo [11]; ORCiD logo [1] « less
  1. Univ. of Nebraska, Lincoln, NE (United States)
  2. Friedrich-Alexander Univ., Erlangen (Germany)
  3. Stanford Univ., CA (United States); SLAC National Accelerator Lab., Menlo Park, CA (United States)
  4. Univ. of Puerto Rico, Mayaguez (Puerto Rico)
  5. SLAC National Accelerator Lab., Menlo Park, CA (United States). Linac Coherent Light Source (LCLS)
  6. SLAC National Accelerator Lab., Menlo Park, CA (United States). Linac Coherent Light Source (LCLS) and Photon Ultrafast Laser Science and Engineering Inst. (PULSE)
  7. SLAC National Accelerator Lab., Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL)
  8. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  9. Univ. of California, San Francisco, CA (United States)
  10. Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
  11. SLAC National Accelerator Lab., Menlo Park, CA (United States); Univ. of California, San Francisco, CA (United States)
Publication Date:
Research Org.:
SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL) and Linac Coherent Light Source (LCLS); Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22). 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); German Research Foundation (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
OSTI Identifier:
1604575
Grant/Contract Number:  
AC02-76SF00515; AC02-06CH11357; GM117126; STC-1231306; GM123159; GM124149; F32 HL129989; 17-SC-20-SC; NIH GM123159; R24GM111072
Resource Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 116; Journal Issue: 51; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; X-ray crystallography; cysteine modification; enzyme conformational; dynamics; XFEL; radiation damage

Citation Formats

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, and Wilson, Mark A. Mix-and-inject XFEL crystallography reveals gated conformational dynamics during enzyme catalysis. United States: N. p., 2019. Web. doi:10.1073/pnas.1901864116.
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. Mix-and-inject XFEL crystallography reveals gated conformational dynamics during enzyme catalysis. United States. doi:https://doi.org/10.1073/pnas.1901864116
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, and Wilson, Mark A. Wed . "Mix-and-inject XFEL crystallography reveals gated conformational dynamics during enzyme catalysis". United States. doi:https://doi.org/10.1073/pnas.1901864116. https://www.osti.gov/servlets/purl/1604575.
@article{osti_1604575,
title = {Mix-and-inject XFEL crystallography reveals gated conformational dynamics during enzyme catalysis},
author = {Dasgupta, Medhanjali and Budday, Dominik and de Oliveira, Saulo H. P. and Madzelan, Peter and Marchany-Rivera, Darya and Seravalli, Javier and Hayes, Brandon and Sierra, Raymond G. and Boutet, Sébastien and Hunter, Mark S. and Alonso-Mori, Roberto and Batyuk, Alexander and Wierman, Jennifer and Lyubimov, Artem and Brewster, Aaron S. and Sauter, Nicholas K. and Applegate, Gregory A. and Tiwari, Virendra K. and Berkowitz, David B. and Thompson, Michael C. and Cohen, Aina E. and Fraser, James S. and Wall, Michael E. and van den Bedem, Henry and Wilson, Mark A.},
abstractNote = {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.},
doi = {10.1073/pnas.1901864116},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 51,
volume = 116,
place = {United States},
year = {2019},
month = {12}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 2 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Is one solution good enough?
journal, March 2006

  • Furnham, Nicholas; Blundell, Tom L.; DePristo, Mark A.
  • Nature Structural & Molecular Biology, Vol. 13, Issue 3
  • DOI: 10.1038/nsmb0306-184

Serial femtosecond crystallography: A revolution in structural biology
journal, July 2016

  • Martin-Garcia, Jose M.; Conrad, Chelsie E.; Coe, Jesse
  • Archives of Biochemistry and Biophysics, Vol. 602
  • DOI: 10.1016/j.abb.2016.03.036

Evolution of New Enzymatic Function by Structural Modulation of Cysteine Reactivity in Pseudomonas fluorescens Isocyanide Hydratase
journal, July 2010

  • Lakshminarasimhan, Mahadevan; Madzelan, Peter; Nan, Ruth
  • Journal of Biological Chemistry, Vol. 285, Issue 38
  • DOI: 10.1074/jbc.M110.147934

Integrative, dynamic structural biology at atomic resolution—it's about time
journal, March 2015

  • van den Bedem, Henry; Fraser, James S.
  • Nature Methods, Vol. 12, Issue 4
  • DOI: 10.1038/nmeth.3324

Coot model-building tools for molecular graphics
journal, November 2004

  • Emsley, Paul; Cowtan, Kevin
  • Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12, p. 2126-2132
  • DOI: 10.1107/S0907444904019158

Direct observation of ultrafast large-scale dynamics of an enzyme under turnover conditions
journal, March 2018

  • Aviram, Haim Yuval; Pirchi, Menahem; Mazal, Hisham
  • Proceedings of the National Academy of Sciences, Vol. 115, Issue 13
  • DOI: 10.1073/pnas.1720448115

Discovery of a Novel Enzyme, Isonitrile Hydratase, Involved in Nitrogen-Carbon Triple Bond Cleavage
journal, April 2001

  • Goda, Masahiko; Hashimoto, Yoshiteru; Shimizu, Sakayu
  • Journal of Biological Chemistry, Vol. 276, Issue 26
  • DOI: 10.1074/jbc.M007856200

Structural and dynamical description of the enzymatic reaction of a phosphohexomutase
journal, March 2019

  • Stiers, Kyle M.; Graham, Abigail C.; Zhu, Jian-She
  • Structural Dynamics, Vol. 6, Issue 2
  • DOI: 10.1063/1.5092803

Conformational Gating of Electron Transfer from the Nitrogenase Fe Protein to MoFe Protein
journal, May 2010

  • Danyal, Karamatullah; Mayweather, Diana; Dean, Dennis R.
  • Journal of the American Chemical Society, Vol. 132, Issue 20
  • DOI: 10.1021/ja101737f

The role of dimer asymmetry and protomer dynamics in enzyme catalysis
journal, January 2017


Time-resolved crystallography reveals allosteric communication aligned with molecular breathing
journal, September 2019


Structural heterogeneity in protein crystals
journal, September 1986

  • Smith, Janet L.; Hendrickson, Wayne A.; Honzatko, Richard B.
  • Biochemistry, Vol. 25, Issue 18
  • DOI: 10.1021/bi00366a008

Modeling discrete heterogeneity in X-ray diffraction data by fitting multi-conformers
journal, September 2009

  • van den Bedem, Henry; Dhanik, Ankur; Latombe, Jean-Claude
  • Acta Crystallographica Section D Biological Crystallography, Vol. 65, Issue 10
  • DOI: 10.1107/S0907444909030613

Structure-based design of targeted covalent inhibitors
journal, January 2018

  • Lonsdale, Richard; Ward, Richard A.
  • Chemical Society Reviews, Vol. 47, Issue 11
  • DOI: 10.1039/C7CS00220C

qFit-ligand Reveals Widespread Conformational Heterogeneity of Drug-Like Molecules in X-Ray Electron Density Maps
journal, November 2018

  • van Zundert, Gydo C. P.; Hudson, Brandi M.; de Oliveira, Saulo H. P.
  • Journal of Medicinal Chemistry, Vol. 61, Issue 24
  • DOI: 10.1021/acs.jmedchem.8b01292

Kinematic Flexibility Analysis: Hydrogen Bonding Patterns Impart a Spatial Hierarchy of Protein Motion
journal, September 2018

  • Budday, Dominik; Leyendecker, Sigrid; van den Bedem, Henry
  • Journal of Chemical Information and Modeling, Vol. 58, Issue 10
  • DOI: 10.1021/acs.jcim.8b00267

PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010

  • Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221
  • DOI: 10.1107/S0907444909052925

Efficient coupling of catalysis and dynamics in the E1 component of Escherichia coli pyruvate dehydrogenase multienzyme complex
journal, January 2008

  • Kale, S.; Ulas, G.; Song, J.
  • Proceedings of the National Academy of Sciences, Vol. 105, Issue 4
  • DOI: 10.1073/pnas.0709328105

Expanding the functional diversity of proteins through cysteine oxidation
journal, December 2008


Bringing diffuse X-ray scattering into focus
journal, June 2018

  • Wall, Michael E.; Wolff, Alexander M.; Fraser, James S.
  • Current Opinion in Structural Biology, Vol. 50
  • DOI: 10.1016/j.sbi.2018.01.009

Automated identification of functional dynamic contact networks from X-ray crystallography
journal, August 2013

  • van den Bedem, Henry; Bhabha, Gira; Yang, Kun
  • Nature Methods, Vol. 10, Issue 9
  • DOI: 10.1038/nmeth.2592

Diverse Functional Roles of Reactive Cysteines
journal, November 2012

  • Pace, Nicholas J.; Weerapana, Eranthie
  • ACS Chemical Biology, Vol. 8, Issue 2
  • DOI: 10.1021/cb3005269

Modelling dynamics in protein crystal structures by ensemble refinement
journal, December 2012


Multiple Conformational Changes in Enzyme Catalysis
journal, July 2002


Diffraction before destruction
journal, July 2014

  • Chapman, Henry N.; Caleman, Carl; Timneanu, Nicusor
  • Philosophical Transactions of the Royal Society B: Biological Sciences, Vol. 369, Issue 1647
  • DOI: 10.1098/rstb.2013.0313

Accessing protein conformational ensembles using room-temperature X-ray crystallography
journal, September 2011

  • Fraser, J. S.; van den Bedem, H.; Samelson, A. J.
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue 39
  • DOI: 10.1073/pnas.1111325108

Native proteins trap high-energy transit conformations
journal, October 2015


The 1.0 Å crystal structure of Ca2+-bound calmodulin: an analysis of disorder and implications for functionally relevant plasticity
journal, September 2000

  • Wilson, Mark A.; Brunger, Axel T.
  • Journal of Molecular Biology, Vol. 301, Issue 5
  • DOI: 10.1006/jmbi.2000.4029

Intrinsic motions along an enzymatic reaction trajectory
journal, November 2007

  • Henzler-Wildman, Katherine A.; Thai, Vu; Lei, Ming
  • Nature, Vol. 450, Issue 7171
  • DOI: 10.1038/nature06410