Unmasking the Conformational Stability and Inhibitor Binding to SARS-CoV-2 Main Protease Active Site Mutants and Miniprecursor.

Autor: Kovalevsky A; Neutron Scattering Division, Oak Ridge National Laboratory, 1 Bethel Valley Road, Oak Ridge, TN 37831, USA. Electronic address: kovalevskyay@ornl.gov., Coates L; Second Target Station, Oak Ridge National Laboratory, 1 Bethel Valley Road, Oak Ridge, TN 37831, USA., Kneller DW; Neutron Scattering Division, Oak Ridge National Laboratory, 1 Bethel Valley Road, Oak Ridge, TN 37831, USA. Electronic address: https://www.twitter.com/DanielKneller., Ghirlando R; Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHS, Bethesda, MD 20892-0520, USA., Aniana A; Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHS, Bethesda, MD 20892-0520, USA., Nashed NT; Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHS, Bethesda, MD 20892-0520, USA., Louis JM; Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHS, Bethesda, MD 20892-0520, USA. Electronic address: johnl@niddk.nih.gov.
Jazyk: angličtina
Zdroj: Journal of molecular biology [J Mol Biol] 2022 Dec 30; Vol. 434 (24), pp. 167876. Date of Electronic Publication: 2022 Nov 02.
DOI: 10.1016/j.jmb.2022.167876
Abstrakt: We recently demonstrated that inhibitor binding reorganizes the oxyanion loop of a monomeric catalytic domain of SARS CoV-2 main protease (MPro) from an unwound (E) to a wound (active, E*) conformation, independent of dimerization. Here we assess the effect of the flanking N-terminal residues, to imitate the MPro precursor prior to its autoprocessing, on conformational equilibria rendering stability and inhibitor binding. Thermal denaturation (T m ) of C145A mutant, unlike H41A, increases by 6.8 °C, relative to wild-type mature dimer. An inactivating H41A mutation to maintain a miniprecursor containing TSAVL[Q or E] of the flanking nsp4 sequence in an intact form [ (-6) MPro H41A and (-6*) MPro H41A , respectively], and its corresponding mature MPro H41A were systematically examined. While the H41A mutation exerts negligible effect on T m and dimer dissociation constant (K dimer ) of MPro H41A , relative to the wild type MPro, both miniprecursors show a 4-5 °C decrease in T m and > 85-fold increase in K dimer as compared to MPro H41A . The K d for the binding of the covalent inhibitor GC373 to (-6*) MPro H41A increases ∼12-fold, relative to MPro H41A , concomitant with its dimerization. While the inhibitor-free dimer exhibits a state in transit from E to E* with a conformational asymmetry of the protomers' oxyanion loops and helical domains, inhibitor binding restores the asymmetry to mature-like oxyanion loop conformations (E*) but not of the helical domains. Disorder of the terminal residues 1-2 and 302-306 observed in both structures suggest that N-terminal autoprocessing is tightly coupled to the E-E* equilibrium and stable dimer formation.
Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Published by Elsevier Ltd.)
Databáze: MEDLINE
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