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 (PLpro) 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 down arrow X motif in pp1a/1ab is crucial for recognition and cleavage by PLpro. 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 PLpro. The results reveal how the substrate sequence affects the efficiency of PLpro-catalyzed hydrolysis. In particular, a proline at the P2 ' position promotes catalysis, as validated by residue substitutions and mass spectrometry-based analyses. Analysis of PLpro 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 PLpro. The results will help in identifying PLpro substrates and guiding inhibitor design. Computational modeling of how SARS-CoV-2 PLpro binds viral polyprotein-derived oligopeptide substrates reveals that a proline located at the P2 ' position promotes catalysis, validated by residue substitutions and mass spectrometry-based analyses.