Ubiquitin C-terminal hydrolases (UCHs) comprise a family of small ubiquitin-specific proteases of uncertain function. Although no cellular substrates have been identified for UCHs, their highly tissue-specific expression patterns and the association of UCH-L1 mutations with human disease strongly suggest a critical role. The structure of the yeast UCH Yuh1-ubiquitin aldehyde complex identified an active site crossover loop predicted to limit the size of suitable substrates. We report the 1.45 Å resolution crystal structure of human UCH-L3 in complex with the inhibitor ubiquitin vinylmethylester, an inhibitor that forms a covalent adduct with the active site cysteine of ubiquitin-specific proteases. This structure confirms the predicted mechanism of the inhibitor and allows the direct comparison of a UCH family enzyme in the free and ligand-bound state. We also show the efficient hydrolysis by human UCH-L3 of a 13-residue peptide in isopeptide linkage with ubiquitin, consistent with considerable flexibility in UCH substrate size. We propose a model for the catalytic cycle of UCH family members which accounts for the hydrolysis of larger ubiquitin conjugates.A wide variety of cellular biochemical pathways are regulated by the post-translational addition of ubiquitin (Ub) 1 to protein substrates (1, 2). Although the enzymatic process of ubiquitin ligation has been studied extensively, that of ubiquitin deconjugation is less well understood. A group of enzymes collectively termed deubiquitinating enzymes (DUBs) catalyzes the hydrolysis of the isopeptide linkage that joins the C-terminal glycine of ubiquitin and a lysine side chain on the target polypeptide. The DUB family consists of four structurally distinct subfamilies: the ubiquitin C-terminal hydrolases (UCHs), ubiquitin-processing proteases (Ubps, USPs), OTU domaincontaining enzymes (otubains) and the Jab/MPN domain-associated metalloisopeptidase domain-containing metalloproteases (3, 4). The first three enzyme classes all possess the sequence signature of cysteine proteases: a conserved catalytic triad of cysteine, histidine, and aspartic acid residues. Sequence analysis of the human genome predicts at least 100 DUBs, which begs the question of their physiological roles. Although restricted substrate specificity is predicted to underlie the requirement for such a large enzyme family, little is known about substrate specificity determinants. The recently published structures for the Ubp family member USP7 (HAUSP) in the unliganded and liganded state (5) affords a unique opportunity to examine specificity determinants for this enzyme. Although the HAUSP catalytic residues are misaligned in the unliganded state, the catalytic core undergoes a dramatic conformational change when in a complex with the inhibitor ubiquitin aldehyde (Ubal), resulting in alignment of the catalytic residues with the C terminus of Ub. The open configuration of the HAUSP active site explains the ability of this class of enzyme to accommodate large ubiquitin conjugates as substrates (e.g....

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There's always a catch. The post‐translational modification of proteins with ubiquitin (Ub) or ubiquitin‐like (Ubl) modifiers is an important signal in the regulation of a variety of biological processes, such as degradation and regulation of gene expression. Here we report the synthesis of a panel of peptide vinyl sulfones (see scheme) harboring various portions of the Ub C terminus by using a safety‐catch linker. Depending on their length, such compounds can efficiently target Ubl‐specific proteases.