Sulfhydryl signals were shown to be readily observable in nuclear Overhauser enhancement spectra of perdeuterated proteins, even for partially solvent-accessible thiol groups. For dimeric SARS-CoV-2 main protease, these signals proved invaluable both for achieving nearly complete NMR backbone assignment in both apo- and substrate-bound states and for validating structural features.
The 68-kDa homodimeric 3C-like protease of SARS-CoV-2, Mpro (3CLpro/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 Mpro (Mpro
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 Mpro
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 Mpro, its substrate peptides, and other ligands, which present opportunities for competitive binding studies in future drug design efforts.Zum Volltext