An in-solution snapshot of SARS-COV-2 main protease maturation process and inhibition.

Abstract

The main protease from SARS-CoV-2 (M^pro) is responsible for cleavage of the viral polyprotein. M^pro self-processing is called maturation, and it is crucial for enzyme dimerization and activity. Here we use C145S M^pro to study the structure and dynamics of N-terminal cleavage in solution. Native mass spectroscopy analysis shows that mixed oligomeric states are composed of cleaved and uncleaved particles, indicating that N-terminal processing is not critical for dimerization. A 3.5 Å cryo-EM structure provides details of M^pro N-terminal cleavage outside the constrains of crystal environment. We show that different classes of inhibitors shift the balance between oligomeric states. While non-covalent inhibitor MAT-POS-e194df51-1 prevents dimerization, the covalent inhibitor nirmatrelvir induces the conversion of monomers into dimers, even with intact N-termini. Our data indicates that the M^pro dimerization is triggered by induced fit due to covalent linkage during substrate processing rather than the N-terminal processing.