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.
Keywords: AlphaFold-Multimers; Mpro/3CLpro; NMR spectroscopy; SARS-CoV-2; ligand binding.
【저자키워드】 SARS-CoV-2, NMR spectroscopy, Mpro/3CLPro, ligand binding., AlphaFold-Multimers, 【초록키워드】 drug design, variant, 3CL pro, protease, Protein, peptides, experiment, NMR, binding, Combination, Ligand, Interaction, High-resolution, ligands, M pro, sequence, competitive binding, solvent, backbone, aliphatic, N-terminal, protons, antiviral drug target, effective, amide, identify, remained, reveal, homodimeric, thiol group, 【제목키워드】 AID, NMR, signal, sulfhydryl,