Biology subjects

Wall, M. E.

Publications and source records attributed to Wall, M. E..

5 recordsLinked to original sources

bioRxiv2023

Changes in an Enzyme Ensemble During Catalysis Observed by High Resolution XFEL Crystallography

Enzymes populate ensembles of structures with intrinsically different catalytic proficiencies that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL) to observe catalysis in a designed mutant (G150T) isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations and formation of the thioimidate catalytic intermediate selects for catalytically competent substates. A prior proposal for active site cysteine charge-coupled conformational changes in ICH is validated by determining structures of the enzyme over a range of pH values. A combination of large molecular dynamics simulations of the enzyme in crystallo and time-resolved electron density maps shows that ionization of the general acid Asp17 during catalysis causes additional conformational changes that propagate across the dimer interface, connecting the two active sites. These ionization-linked changes in the ICH conformational ensemble permit water to enter the active site in a location that is poised for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

biochemistry↗

bioRxiv2022

Molecular-Dynamics Simulation Methods for Macromolecular Crystallography

To assess the potential benefits of molecular-dynamics (MD) simulations for macromolecular crystallography (MX), we performed room-temperature X-ray diffraction studies of the catalytic subunit of mouse protein kinase A (PKA-C). We then performed crystalline MD simulations of PKA-C, computed simulated electron densities from the water, protein, and ion components of the MD simulations, and carefully compared them to the initial crystal structure. The results led to the development of an MD-MX analysis procedure and several associated methods: 1) density comparison to evaluate consistency between the MD and the initial crystal structure model; 2) water building to generate alternative solvent models; and 3) protein remodeling to improve the crystal structure where interpretation of density is unclear. This procedure produced a revised structure of PKA with a new ordered water model and a modified protein structure. The revisions yield new insights into PKA mechanisms, including: a sensitivity of the His294 conformation to protonation state, with potential consequences for regulation of substrate binding; a remodeling of the Lys217 side chain along with a bound phosphate; an alternative conformation for Lys213 associated with binding to the regulatory subunit; and an alternative conformation for catalytic base Asp166 and nearby waters, suggesting a mechanism of progression of the phosphotransfer reaction via changes in Mg2+ coordination. Based on the benefits seen applying these methods to PKA, we recommend incorporating our MD-MX procedure into MX studies, to decide among ambiguous interpretations of electron density that occur, inevitably, as part of standard model refinement.

biophysics↗

bioRxiv2021

Enhancing Sampling of Water Rearrangements on Ligand Binding: A Comparison of Techniques

1Water often plays a key role in protein structure, molecular recognition, and mediating protein-ligand interactions. Thus, free energy calculations must adequately sample water motions, which often proves challenging in typical MD simulation timescales. Thus, the accuracy of methods relying on MD simulations ends up limited by slow water sampling. Particularly, as a ligand is removed or modified, bulk water may not have time to fill or rearrange in the binding site. In this work, we focus on several molecular dynamics (MD) simulation-based methods attempting to help rehydrate buried water sites: BLUES, using nonequilibrium candidate Monte Carlo (NCMC); grand, using grand canonical Monte Carlo (GCMC); and normal MD. We assess the accuracy and efficiency of these methods in rehydrating target water sites. We selected a range of systems with varying numbers of waters in the binding site, as well as those where water occupancy is coupled to the identity or binding mode of the ligand. We analyzed rehydration of buried water sites in binding pockets using both clustering of trajectories and direct analysis of electron density maps. Our results suggest both BLUES and grand enhance water sampling relative to normal MD and grand is more robust than BLUES, but also that water sampling remains a major challenge for all of the methods tested. The lessons we learned for these methods and systems are discussed.

biophysics↗

bioRxiv2021

Reproducibility of protein X-ray diffuse scattering and potential utility for modeling atomic displacement parameters

Protein structure and dynamics can be probed using X-ray crystallography. Whereas the Bragg peaks are only sensitive to the average unit-cell electron density, the signal between the Bragg peaks -- diffuse scattering -- is sensitive to spatial correlations in electron-density variations. Although diffuse scattering contains valuable information about protein dynamics, the diffuse signal is more difficult to isolate from the background compared to the Bragg signal, and the reproducibility of diffuse signal is not yet well understood. We present a systematic study of the reproducibility of diffuse scattering from isocyanide hydratase (ICH) in three different protein forms. Both replicate diffuse datasets and datasets obtained from different mutants were similar in pairwise comparisons (Pearson correlation coefficient (CC) [≥]0.8). The data were processed in a manner inspired by previously published methods using custom software with modular design, enabling us to perform an analysis of various data processing choices to determine how to obtain the highest quality data as assessed using unbiased measures of symmetry and reproducibility. The diffuse data then were used to characterize atomic mobility using a liquid-like motions (LLM) model. This characterization was able to discriminate between distinct anisotropic atomic displacement parameter (ADP) models arising from different anisotropic scaling choices that agreed comparably with the Bragg data. Our results emphasize the importance of data reproducibility as a model-free measure of diffuse data quality, illustrate the ability of LLM analysis of diffuse scattering to select among alternative ADP models, and offer insights into the design of successful diffuse scattering experiments.

biophysics↗

bioRxiv2019

Cysteine modification can gate non-equilibrium conformational dynamics during enzyme catalysis

Summary ParagraphProtein 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 enzymes 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.

biochemistry↗