NMR Observation of Sulfhydryl Signals in SARS‐CoV‐2 Main Protease Aids Structural Studies. (7th September 2022)

• Record Type: Journal Article
• Authors: Robertson, Angus J.; Ying, Jinfa; Bax, Ad
• Is Part Of: Chembiochem. Volume 23:Number 19(2022)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2022-09-07
• Subjects: AlphaFold-Multimers -- ligand binding -- NMR spectroscopy -- Mpro/3CLpro -- SARS-CoV-2; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.202200471
• Languages: English
• ISSNs: 1439-4227

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. Abstract : 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.