Structural Plasticity of the SARS-CoV-2 3CL Mpro Active Site Cavity Revealed by Room Temperature X-ray Crystallography

Abstract

The COVID-19 disease caused by the SARS-CoV-2 Coronavirus has become a pandemic health crisis. An attractive target for antiviral inhibitors is the main protease 3CL Mpro due to its essential role in processing the polyproteins translated from viral RNA. Here we report the room temperature X-ray structure of unliganded SARS-CoV-2 3CL Mpro, revealing the resting structure of the active site and the conformation of the catalytic site cavity. Comparison with previously reported low-temperature ligand-free and inhibitor-bound structures suggest that the room temperature structure may provide more relevant information at physiological temperatures for aiding in molecular docking studies.

Room-temperature X-ray crystallography reveals the oxidation and reactivity of cysteine residues in SARS-CoV-2 3CL Mpro: insights into enzyme mechanism and drug design

“…S1 and S2). Such fresh crystals normally did not exhibit an oxidized Cys145, as seen in our previously reported room-temperature structure of the ligandfree enzyme (PDB entry 6wqf; Kneller et al, 2020). Importantly, we could also reproduce structure I by letting the enzyme crystals grow for more than one week at 14 C, thus indicating that the 0.5 mM TCEP reducing agent present in the crystallization drops does not prevent Cys145 oxidation over a prolonged duration.…”

“…The 3CL M pro (Nsp5 M pro ) from SARS CoV-2 was cloned into the pMCSG81-Delta238 plasmid, designated pCSGID-Mpro, expressed and purified according to the published procedure (Kneller et al, 2020). To generate the authentic protease, the N-terminus is flanked by the maltose-binding protein followed by the 3CL M pro autocleavage site SAVLQ#SGFRK (the arrow indicates the cleavage site) corresponding to cleavage between NSP4 and NSP5 in the viral polyprotein.…”

“…The diffraction data were then reduced and scaled using AIMLESS (Evans & Murshudov, 2013) from the CCP4 suite (Winn et al, 2011). Molecular replacement using PDB entry 6wqf (Kneller et al, 2020) was then performed with MOLREP (Vagin & Teplyakov, 2010) from the CCP4 suite. Refinement of each protein structure was conducted using phenix.refine from the Phenix suite of programs (Liebschner et al, 2019) and the Coot molecular-graphics program (Emsley et al, 2010).…”

Room-temperature neutron and X-ray data collection of 3CL Mprofrom SARS-CoV-2

“…It performs a critical function by cleaving the two large viral polyproteins pp1a and pp1ab into smaller functional proteins (Gorbalenya & Snijder, 1996). The lack of a homologous human protein makes 3CL M pro one of the most promising targets for the design of specific protease inhibitors (Zhang et al, 2020;Kneller et al, 2020;Jin et al, 2020). The global crystallographic community has rapidly been exploring chemical space in the search for drug candidates that can block the action of 3CL M pro (Zhang et al, 2020).…”

“…To date, no FDA-approved inhibitors (Pillaiyar et al, 2016) have been developed against the closely related 3CL M pro from the 2003 SARS-CoV that shares 96% amino-acid homology with the 3CL M pro from SARS-CoV-2. 3CL M pro is active as a homodimer that is composed of two amino-acid chains of 306 amino acids (Kneller et al, 2020;Zhou et al, 2020;Jin et al, 2020). Each chain is folded into three domains.…”