Protein structural heterogeneity: A hypothesis for the basis of proteolytic recognition by the main protease of SARS-CoV and SARS-CoV-2

The main protease (M(pro)) of SARS-CoV and SARS-CoV-2 is a key enzyme in viral replication and a promising target for the development of antiviral therapeutics. The understanding of this protein is based on a number of observations derived from earlier x-ray structures, which mostly consider substrates or ligands as the main reason behind modulation of the active site. This lead to the concept of substrate-induced subsite cooperativity as an initial attempt to explain the dual binding specificity of this enzyme in recognizing the cleavage sequences at its N- and C-termini, which are important processing steps in obtaining the mature protease. The presented hypothesis proposes that structural heterogeneity is a property of the enzyme, independent of the presence of a substrate or ligand. Indeed, the analysis of M(pro) structures of SARS-CoV and SARS-CoV-2 reveals a conformational diversity for the catalytically competent state in ligand-free structures. Variation in the binding site appears to result from flexibility at residues lining the S(1) subpocket and segments incorporating methionine 49 and glutamine 189. The structural evidence introduces “structure-based recognition” as a new paradigm in substrate proteolysis by M(pro). In this concept, the binding space in subpockets of the enzyme varies in a non-cooperative manner, causing distinct conformations, which recognize and process different cleavage sites, as the N- and C-termini. Insights into the recognition basis of the protease provide explanation to the ordered processing of cleavage sites. The hypothesis expands the conformational space of the enzyme and consequently opportunities for drug development and repurposing efforts.