Structural Divergence without Functional Impact: Comparative Characterization of SARS-CoV-2 3CL-Mpro Variants Using Cleavage Site Substrates
Saha, D.; Dakhili, S. Y. T.; Mar, E.; Chen, Y. S.; VanPetegem, F.; Bromme, D.
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SARS-CoV-2 3CL-Mpro is essential for viral replication. Several circulating variants carry mutations distant from the catalytic residues yet maintain proteolytic function. The Omicron substitution P132H exemplifies this tolerance. However, preservation of overall activity does not exclude subtler effects on substrate recognition or selectivity, which can be influenced by distal structural perturbations. To define whether this mutation alters enzymatic activity at a cleavage junction distinct from the Nsp4-Nsp5 site, we measured and compared the biochemical properties of Wuhan with Omicron (e.g. steady-state kinetics) and determined a high-resolution structure of the Omicron (P132H) 3CL-Mpro in complex with an Nsp8-Nsp9 peptide. Catalytic efficiency and Nirmatrelvir sensitivity were comparable to wild type across both cleavage-site substrates, and all variants retained the characteristic pH optimum. In contrast, P132H displayed reduced thermal stability at elevated temperature. Crystallographic analysis showed that His132 adopts a distinct conformation upon substrate binding that reorganizes interactions with Glu240 and neighboring residues while preserving active-site geometry. Together, these results define how 3CL-Mpro tolerates variant-associated mutations without compromising activity at a noncanonical cleavage junction.
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