NMR Observation of Sulfhydryl Signals in SARS-CoV-2 Main Protease Aids Structural Studies
(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.
(Less)
- author
- Robertson, Angus J. LU ; Ying, Jinfa and Bax, Ad
- organization
- publishing date
- 2022
- 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
-
- scopus:85137536129
- pmid:35972230
- 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
- 2024-10-18 03:29:22
@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 (>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}}, }