Studies on the selectivity of the SARS-CoV-2 papain-like protease reveal the importance of the P2' proline of the viral polyprotein.

The SARS-CoV-2 papain-like protease (PL pro ) is an antiviral drug target that catalyzes the hydrolysis of the viral polyproteins pp1a/1ab, so releasing the non-structural proteins (nsps) 1-3 that are essential for the coronavirus lifecycle. The LXGG↓X motif in pp1a/1ab is crucial for recognition and cleavage by PL pro . We describe molecular dynamics, docking, and quantum mechanics/molecular mechanics (QM/MM) calculations to investigate how oligopeptide substrates derived from the viral polyprotein bind to PL pro . The results reveal how the substrate sequence affects the efficiency of PL pro -catalyzed hydrolysis. In particular, a proline at the P2' position promotes catalysis, as validated by residue substitutions and mass spectrometry-based analyses. Analysis of PL pro catalyzed hydrolysis of LXGG motif-containing oligopeptides derived from human proteins suggests that factors beyond the LXGG motif and the presence of a proline residue at P2' contribute to catalytic efficiency, possibly reflecting the promiscuity of PL pro . The results will help in identifying PL pro substrates and guiding inhibitor design.