Surprising variety in the USP deubiquitinase catalytic mechanism

Abstract

The USP family of deubiquitinases (DUBs) controls many ubiquitin-dependent signaling events. This generates therapeutic potential, with active-site inhibitors in preclinical and clinical studies. Understanding of the USP active site was so far primarily guided by USP7 data, where the catalytic triad consists of cysteine, histidine and a third residue (first critical residue), which polarizes the histidine through a hydrogen bond. A conserved aspartate (second critical residue) is directly adjacent to this first critical residue. Here we study the roles of these critical residues in a subset of USPs and reveal a remarkable variety in function. While USP7 relies on the first critical residue for catalysis, this residue is dispensable in USP1, USP15, USP40 and USP48. Instead, their second critical residue is vital for catalysis. Interestingly, without their respective vital residue USP7, USP15 and USP40 can still perform nucleophilic attack. The diverging catalytic mechanisms of USP1 and USP7 are independent of substrate and retained in cells for USP1. The unexpected variety of catalytic mechanisms in this well-conserved protein family may generate opportunities for selective targeting of individual USPs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/550302v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1f3fdbeorg.highwire.dtl.DTLVardef@197f836org.highwire.dtl.DTLVardef@28b588org.highwire.dtl.DTLVardef@a62a2b_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisO_LIThe roles of the highly conserved critical residues in USP active sites are poorly understood. Here we show that these two residues have varying importance for catalysis between different USPs. C_LIO_LIExcept for USP7, the majority of USPs does not rely on the canonical third catalytic residue (first critical residue). Instead, the USPs tested rely primarily on the highly conserved second critical residue. C_LIO_LIIn some USPs, either critical residues can accommodate nucleophilic attack (USP7, USP40, USP15). USP1 and USP48 are unable to perform the nucleophilic attack without the second critical residue. C_LI