Crystal structure of a deubiquitinating enzyme (human UCH-L3) at 1.8 Å resolution

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Abstract
Ubiquitin C‐terminal hydrolases catalyze the removal of adducts from the C‐terminus of ubiquitin. We have determined the crystal structure of the recombinant human Ubiquitin C‐terminal Hydrolase (UCH‐L3) by X‐ray crystallography at 1.8 å resolution. The structure is comprised of a central antiparallel β‐sheet flanked on both sides by α‐helices. The β‐sheet and one of the helices resemble the well‐known papain‐like cysteine proteases, with the greatest similarity to cathepsin B. This similarity includes the UCH‐L3 active site catalytic triad of Cys95, His169 and Asp184, and the oxyanion hole residue Gln89. Papain and UCH‐L3 differ, however, in strand and helix connectivity, which in the UCH‐L3 structure includes a disordered 20 residue loop (residues 147‐166) that is positioned over the active site and may function in the definition of substrate specificity. Based upon analogy with inhibitor complexes of the papain‐like enzymes, we propose a model describing the binding of ubiquitin to UCH‐L3. The UCH‐L3 active site cleft appears to be masked in the unliganded structure by two different segments of the enzyme (residues 9‐12 and 90‐94), thus implying a conformational change upon substrate binding and suggesting a mechanism to limit non‐specific hydrolysis.