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

Journal Article · · Journal of Molecular Biology
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  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  2. National Institutes of Health (NIH), Bethesda, MD (United States)
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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 (Tm) 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)MProH41A and (-6*)MProH41A, respectively], and its corresponding mature MProH41A were systematically examined. While the H41A mutation exerts negligible effect on Tm and dimer dissociation constant (Kdimer) of MProH41A, relative to the wild type MPro, both miniprecursors show a 4–5 °C decrease in Tm and > 85-fold increase in Kdimer as compared to MProH41A. The Kd for the binding of the covalent inhibitor GC373 to (-6*)MProH41A increases ~12-fold, relative to MProH41A, 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. Finally, 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.

Research Organization:
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Spallation Neutron Source (SNS); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). High Flux Isotope Reactor (HFIR)
Sponsoring Organization:
USDOE; USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK); National Institutes of Health (NIH)
Grant/Contract Number:
AC05-00OR22725
OSTI ID:
1898984
Journal Information:
Journal of Molecular Biology, Journal Name: Journal of Molecular Biology Journal Issue: 24 Vol. 434; ISSN 0022-2836
Publisher:
ElsevierCopyright Statement
Country of Publication:
United States
Language:
English

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
SARS CoV-2 main protease
conformational stability
inhibitor binding
main protease miniprecursor
monomer-dimer equilibrium
oxyanion loop
room-temperature x-ray crystallography