NMR Observation of Sulfhydryl Signals in SARS‐CoV‐2 Main Protease Aids Structural Studies
Angus J. Robertson, Jinfa Ying, Ad Bax
Abstract
Abstract The 68‐kDa homodimeric 3C‐like protease of SARS‐CoV‐2, M pro (3CL pro /Nsp5), is a key antiviral drug target. NMR spectroscopy of this large system proved challenging and resonance assignments have remained incomplete. Here we present the near‐complete (>97 %) backbone assignments of a C145A variant of M pro (M pro C145A ) both with, and without, the N‐terminal auto‐cleavage substrate sequence, in its native homodimeric state. We also present SILLY (Selective Inversion of thioL and Ligand for NOESY), a simple yet effective pseudo‐3D NMR experiment that utilizes NOEs to identify interactions between Cys‐thiol or aliphatic protons, and their spatially proximate backbone amides in a perdeuterated protein background. High protection against hydrogen exchange is observed for 10 of the 11 thiol groups in M pro C145A , even those that are partially accessible to solvent. A combination of SILLY methods and high‐resolution triple‐resonance NMR experiments reveals site‐specific interactions between M pro , its substrate peptides, and other ligands, which present opportunities for competitive binding studies in future drug design efforts.