Recognition of Lys48-Linked Di-ubiquitin and Deubiquitinating Activities of the SARS Coronavirus Papain-like Protease

Deubiquitinating enzymes (DUBs) recognize and cleave linkage-specific polyubiquitin (polyUb) chains, but mechanisms underlying specificity remain elusive in many cases. The severe acute respiratory syndrome (SARS) coronavirus papain-like protease (PLpro) is a DUB that cleaves ISG15, a two-domain Ub-like protein, and Lys48-linked polyUb chains, releasing diUbLys48 products. To elucidate this specificity, we report the 2.85 Å crystal structure of SARS PLpro bound to a diUbLys48 activity-based probe. SARS PLpro binds diUbLys48 in an extended conformation via two contact sites, S1 and S2, which are proximal and distal to the active site, respectively. We show that specificity for polyUbLys48 chains is predicated on contacts in the S2 site and enhanced by an S1-S1′ preference for a Lys48 linkage across the active site. In contrast, ISG15 specificity is dominated by contacts in the S1 site. Determinants revealed for polyUbLys48 specificity should prove useful in understanding PLpro deubiquitinating activities in coronavirus infections. [Display omitted] •A Lys48 linkage-specific diubiquitin activity-based probe selectively labels SARS PLpro•The structure of a diUbLys48∼SARS PLpro complex reveals an extended di-Ub conformation•S2-S1 and S1-S1′ interactions make SARS PLpro specific for K48-linked polyubiquitin•SARS PLpro recognizes Lys48-linked polyUb chains and ISG15 via distinct manners Békés et al. present a high-resolution crystal structure of a SARS virus PLpro∼diUbLys48 complex that reveals an extended conformation of the Lys48-linked diUb unit and shows the biochemical basis for SARS PLpro’s preference for Lys48-linked polyUb chains.