Understanding Cysteine Chemistry Using Conventional and Serial X-Ray Protein Crystallography.

Proteins that use cysteine residues for catalysis or regulation are widely distributed and intensively studied, with many biomedically important examples. Enzymes where cysteine is a catalytic nucleophile typically generate covalent catalytic intermediates whose structures are important for understanding mechanism and for designing targeted inhibitors. The formation of catalytic intermediates can change enzyme conformational dynamics, sometimes activating protein motions that are important for catalytic turnover. However, these transiently populated intermediate species have been challenging to structurally characterize using traditional crystallographic approaches. This review describes the use and promise of new time-resolved serial crystallographic methods to study cysteine-dependent enzymes, with a focus on the main (M) and papain-like (PL) cysteine proteases of SARS-CoV-2 as well as other examples. We review features of cysteine chemistry that are relevant for the design and execution of time-resolved serial crystallography experiments. In addition, we discuss emerging X-ray techniques such as time-resolved sulfur X-ray spectroscopy that may be able to detect changes in sulfur charge state and covalency during catalysis or regulatory modification. In summary, cysteine-dependent enzymes have features that make them especially attractive targets for new time-resolved serial crystallography approaches, which can reveal both changes to enzyme structure and dynamics during catalysis in crystalline samples.