Crystal structure of the catalytic domain of UCHL5, a proteasome-associated human deubiquitinating enzyme, reveals an unproductive form of the enzyme

Abstract

Ubiquitin carboxy-terminal hydrolase L5 (UCHL5) is a proteasome-associated deubiquitinating enzyme, which, along with RPN11 and USP14, is known to carry out deubiquitination on proteasome. As a member of the ubiquitin carboxy-terminal hydrolase (UCH) family, UCHL5 is unusual because, unlike UCHL1 and UCHL3, it can process polyubiquitin chain. However, it does so only when it is bound to the proteasome; in its free form, it is capable of releasing only relatively small leaving groups from the C-terminus of ubiquitin. Such a behavior might suggest at least two catalytically distinct forms of the enzyme, an apo form incapable of chain processing activity, and a proteasome-induced activated form capable of cleaving polyubiquitin chain. Through the crystal structure analysis of two truncated constructs representing the catalytic domain (UCH domain) of this enzyme, we were able to visualize a state of this enzyme that we interpret as its inactive form, because the catalytic cysteine appears to be in an unproductive orientation. While this work was in progress, the structure of a different construct representing the UCH domain was reported; however, in that work the structure reported was that of an inactive mutant [catalytic Cys to Ala; Nishio K et al. (2009) Biochem Biophys Res Commun 390, 855-860], which precluded the observation that we are reporting here. Additionally, our structures reveal conformationally dynamic parts of the enzyme that may play a role in the structural transition to the more active form.

Figure 1

Progress curve of hydrolysis of ubiquitin-AMC (Ub-AMC) catalyzed by UCHL5 constructs: full-length UCHL5 (filled triangle), UCHL5N240 (open circle), UCHL5N240^C88S (Cys88 to Ser mutant, open square), UCHL5N240^H164N (closed diamond), UCHL5N240^D179N (open triangle).

Figure 2

Ribbon representation of the X-ray structure of the catalytic domain of UCHL5 (UCHL5N240). Secondary structures are labeled. The catalytic triad residues and the oxyanion-stabilizing side chain are shown in stick representation. (B) A different view of the structure of UCHL5N240 obtained by rotating the picture in A by 90 degree around the axis indicated here.

Figure 3

Unproductive form of active-site triad in UCHL5. (A) Active-site residues in UCHL5240N (shown as sticks). Carbon atoms are shown in gray, nitrogen in blue, oxygen in red and sulfur in orange. Electron density (2Fo-Fc map contoured at 1.0 σ) is shown in purple line. (B) Superposition of active-site residues in UCH enzymes. Structures overlaid are that of full-length UCHL5 (gray sticks), UCHL5N240 (gray sticks), UCHL5N228C88S (gray sticks), UCHL3 (cyan sticks), UCHL1 (magenta sticks) and Yuh1 (yellow sticks). Distances indicated correspond to UCHL5N240. (C) Superposition of active-site residues of UCHL5N240 with 15 other cysteine proteases from Merops database. Distances indicated correspond to UCHL5N240. In addition to UCH enzymes, the other cysteine proteases used in the superposition are papain, actinidain, chymopapain, caricain, cathepsinF, cathepsinV, cathepsinX, zingipain, USP7, USP2, USP8 and ataxin-3. (D) Interactions stabilizing the non-canonical orientation of Cys88 side chain observed in the crystal structure of UCHL5N240.

Figure 4

Conformational dynamics in UCHL5. (A) Comparison of structures of UCHL5 constructs. Ribbon representation of backbone superposition of UCHL5N240 (gray), UCHL5N228^C88S (cyan), UCHL5N225^C88A (yellow) and full-length UCHL5 (magenta). Ubiquitin (Ub, green ribbon) is modeled into its binding site on UCHL5. (Inset) Expanded view of helix 6 (α6) as observed in structures of different UCHL5 constructs. (B) Steric clash (indicated as a box) between C-terminus of ubiquitin and a part of the crossover loop folding into a 3₁₀ helix as seen in the X-ray structure of UCHL5N228^C88S. Ubiquitin (shown in green ribbon) is placed in its binding site by modeling. (C) Interactions of side chains (shown as sticks) on the 3₁₀ helix (shown in B clashing with the C-terminus of ubiquitin) with nearby residues in UCHL5N228^C88S. (D) Superposition of X-ray structures of UCHL5N240 (gray ribbon, except α8, which is shown in orange) and full-length UCHL5 (magenta ribbon, except for α8, which is shown in cyan). The arrow indicates the relative displacement (of approximately 45 degree) of α8 between the two structures.