Cysteine modification can gate non-equilibrium conformational dynamics during enzyme catalysis: Supplemental Information
Category
Published on
Type
posted-content
Author
Medhanjali DasGupta and Dominik Budday and Peter Madzelan and Javier Seravalli and Brandon Hayes and Raymond G. Sierra and Roberto Alonso-Mori and Aaron S. Brewster and Nicholas K. Sauter and Gregory A. Applegate and Virendra Tiwari and David B. Berkowitz and Michael C. Thompson and James S. Fraser and Michael E. Wall and Henry van den Bedem and Mark A. Wilson
Citation
DasGupta, M. et al., 2019. Cysteine modification can gate non-equilibrium conformational dynamics during enzyme catalysis: Supplemental Information. Available at: http://dx.doi.org/10.1101/524751.
Abstract
Post-translational modification of cysteine residues can regulate protein function and is essential for catalysis by cysteine-dependent enzymes. Covalent modifications neutralize charge on the reactive cysteine thiolate anion and thus alter the active site electrostatic environment. Although a vast number of enzymes rely on cysteine modification for function, precisely how altered structural and electrostatic states of cysteine affect protein dynamics remains poorly understood. Here we use X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent modification of the active site cysteine residue in isocyanide hydratase (ICH) affects the protein conformational ensemble. ICH exhibits a concerted helical displacement upon cysteine modification that is gated by changes in hydrogen bond strength between the cysteine thiolate and the backbone amide of the highly strained residue Ile152. The mobile helix samples alternative conformations in crystals exposed to synchrotron X-ray radiation due to the X-ray-induced formation of a cysteine-sulfenic acid at the catalytic nucleophile (Cys101-SOH). This oxidized cysteine residue resembles the proposed thioimidate intermediate in ICH catalysis. Neither cysteine modification nor helical disorder were observed in X-ray free electron laser (XFEL) diffraction data. Computer simulations confirm cysteine modification-gated helical motion and show how structural changes allosterically propagate through the ICH dimer. Mutations at a Gly residue (Gly150) that modulate helical mobility reduce the ICH catalytic rate and alter its pre-steady state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. Our results suggest that cysteine modification may be a common and likely underreported means for regulating protein conformational dynamics.
DOI
Funding
NSF-STC Biology with X-ray Lasers (NSF-1231306)
