A Crystallographic Snapshot of SARS-CoV-2 Main Protease Maturation Process

Abstract

SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral main protease is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N and C-terminus. Although several mechanisms of self-cleavage had been proposed for SARS-CoV, the lack of structural information for each step is a setback to the understanding of this process. Herein, we used X-ray crystallography to characterize an immature form of the main protease, which revealed major conformational changes in the positioning of domain-three over the active site, hampering the dimerization and diminishing its activity. We propose that this form preludes the cis-cleavage of N-terminal residues within the dimer, leading to the mature active site. Using fragment screening, we probe new cavities in this form which can be used to guide therapeutic development. Furthermore, we characterized a serine site-directed mutant of the main protease bound to its endogenous N and C-terminal residues during the formation of the tetramer. This quaternary form is also present in solution, suggesting a transitional state during the C-terminal trans-cleavage. This data sheds light in the structural modifications of the SARS-CoV-2 main protease during maturation, which can guide the development of new inhibitors targeting its intermediary states.

The temperature-dependent conformational ensemble of SARS-CoV-2 main protease (Mpro)

“…6b ). Interestingly, two small-molecule fragments from recent crystallographic screens 12,29 bind at this area of the dimer interface ( Fig. 6a-b ).…”

Open Science Discovery of Oral Non-Covalent SARS-CoV-2 Main Protease Inhibitors

“…Crystals of Mpro in the monoclinic crystal form (C2), with a single monomer in the asymmetric unit, were grown with drop ratios of 0.15 µl protein, 0.3 µl reservoir solution and 0.05 µl seeds prepared from previously produced crystals of the same crystal form 13 . Crystals in the orthorhombic crystal form (P2 1 2 1 2 1 ), with the Mpro dimer present in the asymmetric unit, were grown with drop ratios of 0.15 µl protein, 0.15 µl reservoir solution and 0.05 µl seeds prepared from crystals of an immature Mpro mutant in the same crystal form 38 .…”

Open Science Discovery of Potent Non-Covalent SARS-CoV-2 Main Protease Inhibitors

“…Crystals of Mpro in the monoclinic crystal form (C2), with a single monomer in the asymmetric unit, were grown with drop ratios of 0.15 µl protein, 0.3 µl reservoir solution and 0.05 µl seeds prepared from previously produced crystals of the same crystal form 15 . Crystals in the orthorhombic crystal form (P212121), with the Mpro dimer present in the asymmetric unit, were grown with drop ratios of 0.15 µl protein, 0.15 µl reservoir solution and 0.05 µl seeds prepared from crystals of an immature Mpro mutant in the same crystal form 44 .…”