Visualizing the Active Site Oxyanion Loop Transition Upon Ensitrelvir Binding and Transient Dimerization of SARS-CoV-2 Main Protease

Kovalevsky, Andrey Y.; Aniana, Annie; Coates, Leighton; Ghirlando, Rodolfo; Nashed, Nashaat T.; Louis, John M.

doi:10.1016/j.jmb.2024.168616

Visualizing the Active Site Oxyanion Loop Transition Upon Ensitrelvir Binding and Transient Dimerization of SARS-CoV-2 Main Protease

Journal Article · Thu May 16 00:00:00 EDT 2024 · Journal of Molecular Biology

DOI:https://doi.org/10.1016/j.jmb.2024.168616· OSTI ID:2372988

^[1]; ^[2]; ^[1]; ^[2]; Nashed, Nashaat T. ^[2]; ^[2]

Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
National Institutes of Health (NIH), Bethesda, MD (United States)

N-terminal autoprocessing from its polyprotein precursor enables creating the mature-like stable dimer interface of SARS-CoV-2 main protease (MPro), concomitant with the active site oxyanion loop equilibrium transitioning to the active conformation (E*) and onset of catalytic activity. Here, through mutagenesis of critical interface residues and evaluating noncovalent inhibitor (ensitrelvir, ESV) facilitated dimerization through its binding to MPro, we demonstrate that residues extending from Ser1 through Glu14 are critical for dimerization. Combined mutations G11A, E290A and R298A (MPro™) restrict dimerization even upon binding of ESV to monomeric MPro™ with an inhibitor dissociation constant of 7.4 ± 1.6 µM. Contrasting the covalent inhibitor NMV or GC373 binding to monomeric MPro, ESV binding enabled capturing the transition of the oxyanion loop conformations in the absence of a reactive warhead and independent of dimerization. Characterization of complexes by room-temperature X-ray crystallography reveals ESV bound to the E* state of monomeric MPro as well as an intermediate approaching the inactive state (E). It appears that the E* to E equilibrium shift occurs initially from G138-F140 residues, leading to the unwinding of the loop and formation of the 3₁₀-helix. Finally, we describe a transient dimer structure of the MPro precursor held together through interactions of residues A5-G11 with distinct states of the active sites, E and E*, likely representing an intermediate in the autoprocessing pathway.

View Accepted Manuscript (DOE)

Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). High Flux Isotope Reactor (HFIR); Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). Spallation Neutron Source (SNS)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 2372988

Journal Information:: Journal of Molecular Biology, Journal Name: Journal of Molecular Biology Journal Issue: 13 Vol. 436; ISSN 0022-2836

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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ensitrelvir binding
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room temperature X-ray crystallography

Visualizing the Active Site Oxyanion Loop Transition Upon Ensitrelvir Binding and Transient Dimerization of SARS-CoV-2 Main Protease

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