Structural basis for catalysis and ubiquitin recognition by theSevere acute respiratory syndrome coronaviruspapain-like protease

Abstract

Papain-like protease (PLpro) is one of two cysteine proteases involved in the proteolytic processing of the polyproteins ofSevere acute respiratory syndrome coronavirus(SARS-CoV). PLproalso shows significantin vitrodeubiquitinating and de-ISGylating activities, although the detailed mechanism is still unclear. Here, the crystal structure of SARS-CoV PLproC112S mutant in complex with ubiquitin (Ub) is reported at 1.4 Å resolution. The Ub core makes mostly hydrophilic interactions with PLpro, while the Leu-Arg-Gly-Gly C-terminus of Ub is located in the catalytic cleft of PLpro, mimicking the P4–P1 residues and providing the first atomic insights into its catalysis. One of the O atoms of the C-terminal Gly residue of Ub is located in the oxyanion hole consisting of the main-chain amides of residues 112 and 113. Mutations of residues in the PLpro–Ub interface lead to reduced catalytic activity, confirming their importance for Ub binding and/or catalysis. The structure also revealed anN-cyclohexyl-2-aminethanesulfonic acid molecule near the catalytic triad, and kinetic studies suggest that this binding site is also used by other PLproinhibitors. Overall, the structure provides a foundation for understanding the molecular basis of coronaviral PLprocatalysis.