Abstract
The SARS-CoV-2 papain-like protease (PL(pro)) and main protease (M(pro)) are nucleophilic cysteine enzymes that catalyze hydrolysis of the viral polyproteins pp1a/1ab. By contrast with M(pro), PL(pro) is also a deubiquitinase (DUB) that accepts post-translationally modified human proteins as substrates. Here we report studies on the DUB activity of PL(pro) using synthetic N(ε)-lysine-branched oligopeptides as substrates that mimic post-translational protein modifications by ubiquitin (Ub) or Ub-like modifiers (UBLs), such as interferon stimulated gene 15 (ISG15). Mass spectrometry (MS)-based assays confirm the DUB activity of isolated recombinant PL(pro). They reveal that the sequence of both the peptide fragment derived from the post-translationally modified protein and that derived from the UBL affects PL(pro) catalysis; the nature of substrate binding in the S sites appears to be more important for catalytic efficiency than binding in the S' sites. Importantly, the results reflect the reported cellular substrate selectivity of PL(pro), i.e. human proteins conjugated to ISG15 are better substrates than those conjugated to Ub or other UBLs. The combined experimental and modelling results imply that PL(pro) catalysis is affected not only by the identity of the substrate residues binding in the S and S' sites, but also by the substrate fold and the conformational dynamics of the blocking loop 2 of the PL(pro):substrate complex. N(ε)-Lysine-branched oligopeptides thus have potential to help the identification of PL(pro) substrates. More generally, the results imply that MS-based assays with N(ε)-lysine-branched oligopeptides have potential to monitor catalysis by human DUBs and hence to inform on their substrate preferences.