A In-solution Snapshot of SARS-COV-2 Main Protease Maturation Process and Inhibition

Abstract Mpro is the main protease of SARS-CoV-2. Its dimeric form is responsible for cleavage of the viral polyprotein at 11 sites, including its own N- and C-termini. Mpro self-cleavage is called maturation, and it is crucial for enzyme dimerization and activity. Recently, we combined x-ray crystallography with biochemical characterization to depict key steps of the maturation process. A single mutant C145S version of Mpro linked to nsp4 cleavage sequence was introduced, allowing us to monitor enzyme shifts between oligomeric states in a human timescale. In here, we used C145S Mpro to study the structure and dynamics of N-terminal cleavage in solution. Native mass spectroscopy analysis showed that mixed oligomeric states are composed of cleaved and uncleaved particles, indicating that N-terminal processing is not critical to oligomerization. A 3.5 Å cryo-EM structure provides details of Mpro N-terminal cleavage in solution, and a glimpse in the dynamic of the active sites outside the constrains of crystal environment. We explored how different classes of inhibitors shift the balance between oligomeric states of Mpro. While non-covalent inhibitor MAT-POS-e194df51-1 prevents oligomerization, we discovered that the covalent inhibitor Nirmatrelvir induces the conversion of monomers into dimers, even with intact N-terminal. Our data indicates that the Mpro dimerization is triggered by the induced fit caused by the covalent linkage during substrate processing rather than the N-terminal processing.