Structure of the ubiquitin hydrolase UCH-L3 complexed with a suicide substrate.
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.
Misaghi
Shahram
S
Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Galardy
Paul J
PJ
Meester
Wim J N
WJ
Ovaa
Huib
H
Ploegh
Hidde L
HL
Gaudet
Rachelle
R
D000595:Amino Acid Sequence; D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D012399:Rotation; D016415:Sequence Alignment; D013379:Substrate Specificity; D025801:Ubiquitin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D043222:Ubiquitin Thiolesterase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D014452:Ubiquitins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*
C482860:UCHL1 protein, human; D025801:Ubiquitin; D014452:Ubiquitins; C496729:ubiquitin vinylmethyl ester; D043222:Ubiquitin Thiolesterase; D003545:Cysteine