CRYSTAL-STRUCTURE OF A DEUBIQUITINATING ENZYME (HUMAN UCH-L3) AT 1.8-ANGSTROM RESOLUTION

Ubiquitin C-terminal hydrolases catalyze the removal of adducts from t he C-terminus of ubiquitin. We have determined the crystal structure o f the recombinant human Ubiquitin C-terminal Hydrolase (UCH-L3) by X-r ay crystallography at 1.8 Angstrom resolution, The structure is compri sed of a central antiparallel beta-sheet flanked on both sides by alph a-helices. The beta-sheet and one of the helices resemble the well-kno wn papain-like cysteine proteases, with the greatest similarity to cat hepsin B. This similarity includes the UCH-L3 active site catalytic tr iad of Cys95, His169 and Asp184, and the oxyanion hole residue Gln89, Papain and UCH-L3 differ, however, in strand and helix connectivity, w hich in the UCH-L3 structure includes a disordered 20 residue loop (re sidues 147-166) that is positioned over the active site and may functi on in the definition of substrate specificity. Based upon analogy with inhibitor complexes of the papain-like enzymes, we propose a model de scribing the binding of ubiquitin to UCH-L3. The UCH-L3 active site cl eft appears to be masked in the unliganded structure by two different segments of the enzyme (residues 9-12 and 90-94), thus implying a conf ormational change upon substrate binding and suggesting a mechanism to limit non-specific hydrolysis.