RT Journal Article
SR Electronic
T1 Mix-and-inject XFEL crystallography reveals gated conformational dynamics during enzyme catalysis
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 524751
DO 10.1101/524751
A1 Medhanjali Dasgupta
A1 Dominik Budday
A1 Saulo H.P. de Oliveira
A1 Peter Madzelan
A1 Darya Marchany-Rivera
A1 Javier Seravalli
A1 Brandon Hayes
A1 Raymond G. Sierra
A1 Sebastian Boutet
A1 Mark Hunter
A1 Roberto Alonso-Mori
A1 Alexander Batyuk
A1 Jennifer Wierman
A1 Artem Lyubimov
A1 Aaron S. Brewster
A1 Nicholas K. Sauter
A1 Gregory A. Applegate
A1 Virendra K. Tiwari
A1 David B. Berkowitz
A1 Michael C. Thompson
A1 Aina Cohen
A1 James S. Fraser
A1 Michael E. Wall
A1 Henry van den Bedem
A1 Mark A. Wilson
YR 2019
UL http://biorxiv.org/content/early/2019/08/24/524751.abstract
AB Summary Paragraph Protein dynamics play an important role in enzyme catalysis1-4. Many enzymes form covalent catalytic intermediates that can alter enzyme structure and conformational dynamics5,6. How these changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question in structural enzymology. Here, we use Mix-and-Inject Serial Femtosecond X-ray Crystallography (MISC) at an X-ray Free Electron Laser (XFEL)7-10, ambient temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent modification of the active site cysteine residue in isocyanide hydratase (ICH) alters the enzyme’s conformational ensemble throughout the catalytic cycle. With MISC, we directly observe formation of a thioimidate covalent intermediate during ICH catalysis. The intermediate exhibits changes in the active site electrostatic environment, disrupting a hydrogen bond and triggering a cascade of conformational changes in ICH. X-ray-induced formation of a cysteine-sulfenic acid at the catalytic nucleophile (Cys101-SOH) with conventional crystallography at ambient temperature induces similar conformational shifts, demonstrating that these enzyme motions result from cysteine modification. Computer simulations show how cysteine modification-gated structural changes allosterically propagate through the ICH dimer. Mutations at 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. Taken together, our results demonstrate the potential of mix-and-inject XFEL crystallography to capture otherwise elusive mechanistic details of enzyme catalysis and dynamics from microcrystalline samples7,11. This approach can connect conformational dynamics to function for the large class of systems that rely on covalently modified cysteine residues for catalysis or regulation, resolving long-standing questions about enzyme mechanism and functionally relevant non-equilibrium enzyme motions.