![]() ![]() SARS-CoV-2 M pro is a functional homodimer of two symmetrically disposed protomers that are each 306 residues in length (33.8 kDa). In addition, increasing evidence shows host cell protein cleavage by viral proteases is a further critical component of viral pathogenicity 6 and recent proteomic analyses have revealed more than 100 substrates cleaved by M pro in human lung and kidney cells including key effectors of transcription, mRNA processing, and translation 7, 8. This is highlighted by the recent success of nirmatrelvir (Paxlovid) in mitigation of serious COVID-19 disease and hospitalization in high-risk patients 5. The functional importance of M pro in the viral life cycle, combined with the absence of closely related homologs in humans and high degree of conservation of M pro and its targets among clinical variants (~96% identical to SARS-CoV-1 M pro) 4, has made the enzyme an attractive target for the development of antiviral drugs. These processing events by M pro represent critical steps prior to subsequent viral assembly and maturation 3. ![]() The main protease of SARS-CoV-2 (M pro, also referred to as 3-chymotrypsin-like protease or 3CLpro) is responsible for the majority of nsp processing, cleaving at 11 conserved sites along the polyprotein including self-excision of M pro by autolytic cleavage of its own N-terminal and C-terminal autoprocessing sites 3. These long polyproteins are processed into 16 smaller functional non-structural proteins (nsps) by two self-encoded cysteine proteases, papain-like protease (nsp3) and the main protease (nsp5). The SARS-CoV-2 genome encodes four structural proteins and two overlapping polyproteins, pp1a and pp1ab, encompassing all the viral proteins required for host invasion and maintenance of the viral lifecycle (Fig. Collectively, these crystallographic snapshots provide valuable mechanistic and structural insights for antiviral therapeutic development. We characterize the underlying noncovalent interactions governing binding and specificity for this diverse set of substrates, showing remarkable plasticity for subsites beyond the anchoring P1(Gln)-P2(Leu/Val/Phe), representing together a near complete analysis of a multiprocessing viral protease. Capture of both acyl-enzyme intermediate and product-like complex forms of a P2(Leu) substrate in the native active site provides direct comparative characterization of these mechanistic steps as well as further informs the basis for enhanced product release of M pro’s own unique C-terminal P2(Phe) cleavage site to prevent autoinhibition. Using C-terminally substituted M pro chimeras, we have determined X-ray crystallographic structures of M pro in complex with 10 of its 11 viral cleavage sites, bound at full occupancy intermolecularly in trans, within the active site of either the native enzyme and/or a catalytic mutant (C145A). Nsp5 is the main protease (M pro) responsible for cleavage at eleven positions along these polyproteins, including at its own N- and C-terminal boundaries, representing essential processing events for viral assembly and maturation. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the pathogen that causes COVID-19, produces polyproteins 1a and 1ab that contain, respectively, 11 or 16 non-structural proteins (nsp). ![]()
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