Unmasking the Conformational Stability and Inhibitor Binding to SARS-CoV-2 Main Protease Active Site Mutants and Miniprecursor. Issue 24 (30th December 2022)

• Record Type: Journal Article
• Authors: Kovalevsky, Andrey; Coates, Leighton; Kneller, Daniel W.; Ghirlando, Rodolfo; Aniana, Annie; Nashed, Nashaat T.; Louis, John M.
• Is Part Of: Journal of molecular biology. Volume 434:Issue 24(2022)
• Publication Date: 2022-12-30
• Subjects: SARS CoV-2 main protease -- monomer–dimer equilibrium -- conformational stability -- inhibitor binding -- room-temperature X-ray crystallography; SARS CoV-2 Severe Acute Respiratory Syndrome CoronaVirus 2 -- COVID-19 COrona VIrus Disease -- nsp non-structural protein -- MPro main protease -- DSF differential scanning fluorimetry -- SEC-MALS size-exclusion chromatography-multiangle light scattering -- SV-AUC sedimentation velocity analytical ultracentrifugation -- CD circular dichroism -- Tm mid-point of thermal denaturation -- Kd inhibitor dissociation constant -- Kdimer dimer dissociation constant -- ΔTm Tm difference; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2022.167876
• Languages: English
• ISSNs: 0022-2836

Abstract

Graphical abstract: Highlights: SARS CoV-2 MPro catalyzes its own cleavage at its termini from polyproteins. Nsp4 residues appended to MPro H41A (miniprecursor) decrease Tm and increase Kdimer . Miniprecursor reveals asymmetry, semi-open conformation, and disorder of termini. Potent inhibitors of mature MPro dimer bind weakly to the miniprecursor. Monomeric active site E conformer presents a strategic target for inhibitor design. Abstract: 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) MPro H41A and (-6*) MPro H41A, respectively], and its corresponding mature MPro H41A were systematically examined. While the H41A mutation exerts negligible effect on Tm and dimer dissociation constant (Kdimer ) of MPro H41A, 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 MPro H41A . The Kd 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.