Catalysis by cysteine-dependent enzymes is required for many essential biochemical pathways, including central metabolism, redox homeostasis, and cellular signaling. Derangements in these pathways occur in many disease states and targeting reactive cysteine residues is an emerging approach for developing potent new drugs. All cysteine-dependent enzymes are transiently modified during catalysis, however little is known about how cysteine modifications alter the structure and functional dynamics of proteins. We will use newly developed time-resolved serial crystallography methods to characterize functionally important non-equilibrium motions in the cysteine-dependent enzyme isocyanide hydratase (ICH) during catalysis. ICH is the principal enzyme that detoxifies isocyanide natural products that possess antibiotic, antiviral, and anticancer properties. Our preliminary data show that transient cysteine modification during ICH catalysis activates a non-equilibrium protein dynamics that can be mapped in atomic detail by mix-and-inject serial X-ray crystallography. The objective of this proposal is to develop and apply new models of catalysis-activated non-equilibrium motions in ICH by analyzing the unprecedentedly information-rich datasets now available from mix-and-inject serial crystallography experiments. We will use serial crystallography and computational approaches to determine how transient modification of the active site cysteine thiolate activates protein motions that involve the whole protein, are asymmetric in the ICH dimer, and are responsible for kinetic heterogeneity in two active sites of the ICH dimer. We have created mutations that alter the equilibrium ICH conformational ensemble, impair catalysis, and diminish the ability of ICH to protect bacteria from isocyanides. Using serial crystallography and enzyme kinetics, we will characterize how these mutations alter allosteric motions during ICH catalysis and prevent efficient intermediate hydrolysis. Finally, we generalize a model of enzyme motions facilitated by conformational strain by determining the role of unusual side-chain and backbone conformational strain in catalysis by a distant ICH homolog that diffracts X-rays to ultrahigh resolution. Combining computation, serial crystallography, and enzyme kinetics, we will determine how conformational strain evolves during ICH catalysis in unprecedented detail. In total, our work will elucidate how catalytic cysteine modification alters conformational ensembles and non-equilibrium motions in enzymes. This work will also drive urgently needed advances in synchrotron serial crystallography methodology in order to dramatically expand the accessibility of these new structural biological techniques.
Enzymes are essential for life but the physical principles dictating their function are still unclear. We will use new methods in time-resolved X-ray crystallography to observe catalysis by an enzyme involved in antibiotic detoxification in real time, opening a new window into this fundamental biochemical process. What is learned from this system will be applicable to a large group of proteins that are subject to similar modifications in health and disease.
