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

J Biol Chem. 2005 Jan 14;280(2):1512-20. doi: 10.1074/jbc.M410770200. Epub 2004 Nov 5.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Crystallization
  • Crystallography, X-Ray
  • Cysteine / metabolism
  • Humans
  • Hydrolysis
  • Models, Biological
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Binding
  • Protein Conformation / drug effects
  • Rotation
  • Sequence Alignment
  • Substrate Specificity
  • Ubiquitin / analogs & derivatives
  • Ubiquitin / chemistry
  • Ubiquitin / metabolism
  • Ubiquitin / pharmacology
  • Ubiquitin Thiolesterase / antagonists & inhibitors
  • Ubiquitin Thiolesterase / chemistry*
  • Ubiquitin Thiolesterase / isolation & purification
  • Ubiquitin Thiolesterase / metabolism*
  • Ubiquitins / chemistry
  • Ubiquitins / isolation & purification
  • Ubiquitins / metabolism*
  • Ubiquitins / pharmacology*


  • UCHL1 protein, human
  • Ubiquitin
  • Ubiquitins
  • ubiquitin vinylmethyl ester
  • Ubiquitin Thiolesterase
  • Cysteine

Associated data

  • PDB/1XD3