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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. 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.

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Publication Date:
Research Org.:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1604575
Grant/Contract Number:  
[AC02-76SF00515; GM117126; STC-1231306; GM123159; F32 HL129989; 17-SC-20-SC]
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
Country of Publication:
United States
Language:
English

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: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:10.1073/pnas.1901864116.
@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. 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}
}

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