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NMR Observation of Sulfhydryl Signals in SARS-CoV-2 Main Protease Aids Structural Studies

Robertson, Angus J. LU ; Ying, Jinfa and Bax, Ad (2022) In ChemBioChem 23(19).
Abstract

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 (MproC145A) 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... (More)

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 (MproC145A) 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 MproC145A, 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.

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author
; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
AlphaFold-Multimers, ligand binding, M/3CLpro, NMR spectroscopy, SARS-CoV-2
in
ChemBioChem
volume
23
issue
19
article number
e202200471
publisher
John Wiley & Sons Inc.
external identifiers
  • pmid:35972230
  • scopus:85137536129
ISSN
1439-4227
DOI
10.1002/cbic.202200471
language
English
LU publication?
yes
id
fcaaa37b-e5a2-4296-936c-5da8e96bfc1c
date added to LUP
2022-10-25 14:49:02
date last changed
2024-06-27 15:06:48
@article{fcaaa37b-e5a2-4296-936c-5da8e96bfc1c,
  abstract     = {{<p>The 68-kDa homodimeric 3C-like protease of SARS-CoV-2, M<sup>pro</sup> (3CL<sup>pro</sup>/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 (&gt;97 %) backbone assignments of a C145A variant of M<sup>pro</sup> (M<sup>pro</sup><sub>C145A</sub>) 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<sup>pro</sup><sub>C145A</sub>, 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<sup>pro</sup>, its substrate peptides, and other ligands, which present opportunities for competitive binding studies in future drug design efforts.</p>}},
  author       = {{Robertson, Angus J. and Ying, Jinfa and Bax, Ad}},
  issn         = {{1439-4227}},
  keywords     = {{AlphaFold-Multimers; ligand binding; M/3CLpro; NMR spectroscopy; SARS-CoV-2}},
  language     = {{eng}},
  number       = {{19}},
  publisher    = {{John Wiley & Sons Inc.}},
  series       = {{ChemBioChem}},
  title        = {{NMR Observation of Sulfhydryl Signals in SARS-CoV-2 Main Protease Aids Structural Studies}},
  url          = {{http://dx.doi.org/10.1002/cbic.202200471}},
  doi          = {{10.1002/cbic.202200471}},
  volume       = {{23}},
  year         = {{2022}},
}