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

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.