Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14

Abstract

The ubiquitin-specific processing protease (UBP) family of deubiquitinating enzymes plays an essential role in numerous cellular processes. Mammalian USP14 (Ubp6 in yeast) is unique among known UBP enzymes in that it is activated catalytically upon specific association with the 26S proteasome. Here, we report the crystal structures of the 45-kDa catalytic domain of USP14 in isolation and in a complex with ubiquitin aldehyde, which reveal distinct structural features. In the absence of ubiquitin binding, the catalytic cleft leading to the active site of USP14 is blocked by two surface loops. Binding by ubiquitin induces a significant conformational change that translocates the two surface loops thereby allowing access of the ubiquitin C-terminus to the active site. These structural observations, in conjunction with biochemical characterization, identify important regulatory mechanisms for USP14.

Figure 1

Structure of the catalytic domain of USP14. (A) Overall structure of the 45-kDa catalytic domain of USP14 (residues 91–494). The structure comprises three domains, Fingers (in green), Palm (in blue), and Thumb (in gold). The active site, comprising the Cys Box (in cyan) and the His Box (in magenta), is located between the Palm and the Thumb. The predicted ubiquitin-binding site is indicated by a black oval circle. The surface representation is shown on the right. Note the absence of the binding groove for the C-terminus of ubiquitin. (B) Comparison of the structures of the catalytic domain between USP14 and HAUSP in a stereo view. USP14 and HAUSP are shown in blue and white, respectively. The active site of free USP14 is covered by two crossover loops BL1 and BL2 (in red). The catalytic Cys residues in USP14 and HAUSP are highlighted in yellow. (C) Comparison of the structures of the catalytic domain between USP14 and Ubp6 in a stereo view. Note that two surface loops (green) in Ubp6 adopt very similar positions as the BL1 and BL2 loops (red) in USP14. All figures were prepared using MOLSCRIPT (Kraulis, 1991) and GRASP (Nicholls et al, 1991).

Figure 2

Sequence alignment of USP14 with its yeast homolog Ubp6 and human HAUSP. Conserved residues are shaded in yellow whereas the catalytic triad residues are highlighted in red. The secondary structural elements above the sequences are indicated for free USP14 (lower) and HAUSP (upper). The four black arrowheads indicate the positions where Cys residues are supposed to be located in a zinc ribbon (Krishna and Grishin, 2004). The coloring scheme for the secondary structural elements of free USP14 is the same as in Figure 1. Sequence alignment employed the program ClustalW. Entries shown are from the SwissProt Database: HAUSP (Human; SW:Q93009); USP14 (Human; SW:P54578); UBP6 (S. cerevisiae; SW:P35127).

Figure 3

The active site of USP14. (A) The 2F_o−F_c electron density at the active site region contoured at 1.8σ. The Cys and His Boxes are colored cyan and magenta, respectively. (B) The catalytic triad residues of USP14 are poised for catalysis. Shown here is a stereo comparison of the active sites of USP14 and HAUSP. The coloring scheme for USP14 is the same as in Figure 1. HAUSP is shown in green. Catalytic triad residues and the oxyanion-coordinating residue are shown. Hydrogen bonds are represented by red dashed lines. (C) The binding cleft for the C-terminus of ubiquitin is blocked by two surface loops in USP14. The binding region for the C-terminus of ubiquitin is shown in two surface representations: solid (left panel) and transparent (right panel). The C-terminus of ubiquitin (green) is placed after superposition of the HAUSP–Ubal structure onto USP14. Several residues of USP14, including Phe331, Tyr333, and Ser432, sterically clash with the C-terminus of ubiquitin. (D) Comparison of the active site conformation in USP14 and Ubp6. Residues from USP14 are labeled, whereas the corresponding residues from Ubp6 are shown in parentheses. The coloring scheme for USP14 is the same as in Figure 1. Ubp6 is shown in gray. Catalytic triad residues and the oxyanion-coordinating residue are shown. Hydrogen bonds are represented by red dashed lines.

Figure 4

Structure of the USP14–Ubal complex. (A) Overall structure of the catalytic core domain of USP14 (91–494, blue) covalently bound to Ubal (in green). The Cys and His Boxes are colored cyan and magenta, respectively. The catalytic Cys114 is shown in a ball-and-stick representation. (B) A large conformational change near the active site induced by Ubal binding. The ubiquitin C-terminus-binding region of USP14 in isolation (in orange) and that in complex with Ubal (in blue) are superimposed and shown in stereo. The C-terminal tail of Ubal is shown in green. Note the conformational changes on the two surface loops, which allow the opening of the binding cleft for the C-terminus of ubiquitin. Amino acids are shown in ball-and-stick representation. (C) Comparison of the conformation of the blocking loops in USP14 and in HAUSP. Two conserved residues from USP14, Phe331 and Tyr333 (Phe409 and Tyr411 in HAUSP), make van der Waals interactions with residues in Ubal. The BL1/BL2 loops and Ubal in the USP14–Ubal complex are colored blue and green, respectively. The HAUSP–Ubal complex is colored gray. The side chains from USP14/HAUSP and Ubal are shown in yellow and orange, respectively.

Figure 5

USP14 interacts with the 19S RP of the 26S proteasome and is activated upon binding. (A) Polyubiquitin chain disassembly by USP14. The proximal end ubiquitin of the triUb chain is fluorescently labeled. As indicated by the sequential appearance of fluorescent bands corresponding to labeled diUb and monoUb, USP14 preferentially cleaves ubiquitin from the distal end of the triUb chain. (B) The Ubl domain of USP14 is responsible for binding to the 19S RP of the 26S proteasome. An approximately equimolar amount of GST-USP14, GST-USP14 (91–494), GST-UBL, or GST (control) was used for each experiment. An equal amount of 26S proteasome or the 19S complex (PA700) was used within the same set of experiments. Only full-length (GST-USP14 (FL)) or the isolated Ubl domain (GST-UBL), but not Ubl-deleted USP14 (GST-Core domain), exhibited binding in GST pull-down assays. Anti-S1 antibody was used to detect 26S proteasome or 19S RP complexes that were bound and then eluted with glutathione (see Materials and methods). (C) USP14 exhibits a significant reactivity with UbVS in the presence of the 19S RP; similar results were obtained with 26S proteasomes (not shown).