Structure of the ubiquitin hydrolase UCH-L3 complexed with a suicide substrate.

Publication Type:

Journal Article

Authors:

Misaghi, Shahram; Galardy, Paul J; Meester, Wim J N; Ovaa, Huib; Ploegh, Hidde L; Gaudet, Rachelle

Source:

J Biol Chem, Volume 280, Issue 2, p.1512-20 (2005)

Keywords:

Amino Acid Sequence, Binding Sites, Crystallization, Crystallography, X-Ray, Cysteine, Humans, Hydrolysis, Models, Biological, Models, Molecular, Molecular Sequence Data, Protein Binding, Protein Conformation, Rotation, Sequence Alignment, Substrate Specificity, Ubiquitin, Ubiquitin Thiolesterase, Ubiquitins

Abstract:

<p>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 A 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.</p>