Structural and functional insights into Ubl domain-mediated regulation of SARS-CoV-2 PLpro

SARS-CoV-2 papain-like protease (PLpro) is essential for viral replication and immune evasion. It contains an N-terminal ubiquitin-like (Ubl) domain, whose involvement in enzymatic function remains poorly understood.

In this study, we investigated the role of the Ubl domain in modulating the structural dynamics and catalytic efficiency of PLpro. Using molecular dynamics (MD) simulations, inhibitor binding assays, and steady-state kinetic analyses, we found that the Ubl domain stabilizes critical structural elements, notably the ridge helix in the thumb subdomain. Removal of the Ubl domain altered substrate processing, reducing catalytic efficiency of the enzyme. Interestingly, free ubiquitin enhanced enzymatic activity, likely via non-canonical binding sites distinct from the SUb1 and SUb2 sites.

These findings uncover a regulatory role for the Ubl domain in allosteric modulation of PLpro activity and reveal additional layers of enzymatic plasticity. Understanding these mechanisms could guide the design of future antiviral therapeutics targeting PLpro's regulatory or allosteric sites.