Animal coronaviruses (CoVs) have been identified to be the origin of Severe Acute Respiratory Syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and probably SARS-CoV-2 that cause severe to fatal diseases in humans. Variations of zoonotic coronaviruses pose potential threats to global human beings. To overcome this problem, we focused on the main protease (M^{pro}), which is an evolutionary conserved viral protein among different coronaviruses. The broad-spectrum anti-coronaviral drug, GC376, was repurposed to target canine coronavirus (CCoV), which causes gastrointestinal infections in dogs. We found that GC376 can efficiently block the protease activity of CCoV M^{pro} and can thermodynamically stabilize its folding. The structure of CCoV M^{pro} in complex with GC376 was subsequently determined at 2.75 Å. GC376 reacts with the catalytic residue C144 of CCoV M^{pro} and forms an (R)- or (S)-configuration of hemithioacetal. A structural comparison of CCoV M^{pro} and other animal CoV M^{pro}s with SARS-CoV-2 M^{pro} revealed three important structural determinants in a substrate-binding pocket that dictate entry and release of substrates. As compared with the conserved A141 of the S1 site and P188 of the S4 site in animal coronaviral M^{pro}s, SARS-CoV-2 M^{pro} contains N142 and Q189 at equivalent positions which are considered to be more catalytically compatible. Furthermore, the conserved loop with residues 46-49 in animal coronaviral M^{pro}s has been replaced by a stable α-helix in SARS-CoV-2 M^{pro}. In addition, the species-specific dimerization interface also influences the catalytic efficiency of CoV M^{pro}s. Conclusively, the structural information of this study provides mechanistic insights into the ligand binding and dimerization of CoV M^{pro}s among different species.
【저자키워드】 COVID-19, SARS-CoV-2, coronavirus, Crystallography, Coronavirus pandemic, X-ray, Protein conformation, viral proteases.,