Abstact

Our previous studies of severe acute respiratory syndrome coronavirus 2 main protease (MPro) precursor monomer indicate that the initial N-terminal nonstructural protein (nsp)4/nsp5 cleavage occurs intramolecularly, with a small fraction of the active site loop equilibrium being in the active state. To understand the influence of dimer formation of MPro upon N-terminal cleavage on the subsequent C-terminal nsp5/nsp6 intermolecular cleavage kinetics, the stepwise processing of a monomeric, inactive precursor containing the native terminal cleavage sites of MPro (MBP-((-6))MPro(C145A(+3))-GB1-6H, 86.2 kDa) by mature WT MPro (MPro(WT)) was investigated. Differential scanning fluorimetry and analytical ultracentrifugation measurements of various MPro constructs suggest that the C145A mutation decreases the dimer dissociation constant (K(dimer)) by approximately 26-fold, relative to WT C145 and H41A. The monomeric precursor's nsp4-nsp5 site appears to saturate MPro(WT)'s active sites and cleave faster, followed by a slower first-order cleavage at the C-terminal site. No detectable product resulting from the C-terminal cleavage is observed until most of the N-terminal cleavage is complete. The initial intermediate product (termed MPro(C145A-IP)) is a homodimer with an estimated K(dimer) of <0.05 muM. In contrast, the first-order kinetics observed for the cleavage of the monomeric form of the intermediate product is at least 300 times faster than that of the dimer form. Room-temperature X-ray structure of the MPro(C145A-IP)-ensitrelvir complex is like that of the MPro(WT)-ensitrelvir complex and reveals a dynamic C-terminal region including MPro residues 302 to 306. These results are interpreted from the point of view of a mechanism in which nsp5-nsp6 cleavage may occur from a monomeric intermediate, and dimer formation restricts this cleavage.