Figure 1.
Overall structure of the OTUB2-Ub complex.
(A) A ribbon diagram showing OTUB2-Ub complex with OTUB2 colored in blue and Ub in red. The active site residues are shown as sticks with carbon atoms colored in cyan; the last two residues of Ub and the covalent linker to Cys51 are drawn as orange sticks. (B) Representative |2Fo-Fc| map contoured at 1σ showing well defined electron density for residues around the active site. (C) Comparison of ligand bound and apo (grey) structures of OTUB2, the large structural changes due to Ub binding are indicated by arrows. (D) Close-up view of the active site of ligand bound and apo OTUB2. (E) Electrostatic surfaces of OTUB2 (bottom panel) and Ub (top panel). Ub is moved upwards and rotated 90° to show the positively charged surface patch that has complementary interactions with OTUB2.
Figure 2.
Key contact areas between OTUB2 and Ub.
(A) Details of interactions between C-terminal tail of Ub and OTUB2. (B) A large contact area formed between the β-sheet of Ub and α8 and α10 helices of OTUB2. (C) Gln40 of Ub is fully buried in the complex interface, making stacking interactions with Tyr195 and triple hydrogen bonds to Asn204 and His206 of OTUB2. (D) Leu73 nests in a hydrophobic pocket formed by residues Ile180, Val193, Tyr195, His206, Phe208, Tyr220 and Tyr225 of OTUB2. The side chains of Ub are colored in cyan and those of OTUB2 in orange.
Figure 3.
Comparison of OTUB2-Ub with other OTU-Ub complexes.
Superposition of OTUB2-Ub (blue and red) with yeast OTU1-Ub (grey) [20] (A), vOTU-Ub (grey) [23] (B) and OTUB1-Ubal-UBC13-Ub (grey / yellow) [13] (C) complexes. The free donor Ub is shown in yellow and the UBC13 is omitted in (C) for clarity.
Figure 4.
Comparison of OTUB2-Ub and OTUB1-Ubal-UBCH5B-Ub complexes.
(A) Overlay of OTUB2-Ub (blue and red) and OTUB1-Ubal-UBCH5B-Ub (grey, grey, cyan and orange respectively) complexes. The N-terminal helix of OTUB1 is shown in magenta for clarity. Side-chains from OTUB1 and the proximal Ub are shown as sticks; black broken lines represent hydrogen bonds, and the red sphere a water molecule. (B) The N-terminal of OTUB2 is shorter and folded to a different direction compared to OTUB1. (C) Close up view of the interactions between the proximal Ub and the OTUB1 α2α3 loop which is two residue shorter in OTUB2 (α1α2 loop).
Figure 5.
OTUB2 has a broader cleavage profile than OTUB1.
(A) Ubiquitin (Ub), Nedd8, ISG15 and SUMO1 were conjugated to the biotinylated peptide VKAKIQD (Ub26–32) as described in [19] and subjected to cleavage by recombinant OTUB2, UCH-L3 or crude cell lysate (- represents untreated control), followed by SDS-PAGE separation and analysis by streptavidin-HRP immunoblotting. (B) di-SUMO2/3 was incubated with DMSO, OTUB2, OTUB2delta, OTUB1 or crude lysate for the indicated times, followed by SDS-PAGE separation and analysis by immunoblotting. (C) Linear di-Ubiquitin (di-Ub) was incubated with OTUB2, UCH-L3 or DMSO for the indicated times, followed by SDS-PAGE separation and analysis by immunoblotting. (D) di-Ub substrates with the linkages Lys6, 11, 27, 29, 33, 48 or 63 were incubated with either wild type (Wt) or catalytically inactive C51S mutant (M) OTUB2 for four hours, followed by SDS-PAGE and immunoblotting analysis.
Figure 6.
OTUB N-terminal tails modulate DUB activity and Ub chain linkage specificity.
(A) Design of OTUB1-(N-term)-OTUB2 (Otub1–2) and OTUB2-(N-term)-OTUB1 (Otub2–1) chimera constructs and recombinant proteins (see also S2 Fig.). (B) Active site labeling using HA-UbBr2 (Br2) or HA-Ub-VME (VME) revealed that the OTUB1 N-terminal tail affects labeling selectivity towards the VME probe. (C) Ub-Rhodamine activity assay revealing the restricting effect of the N-terminal tail of OTUB1 on both, OTUB1 and OTUB2. (D) Magnification of the assay scale shown in (C) to reveal enzymatic activities of the OTUB1 (Otub1, Otub 2–1) proteins. (E) K-values calculated from the Ub-Rhodamine assays (C-D) demonstrating the restricting effect of the OTUB1 N-terminal tail. (F-G) Lys48 and Lys63 tetra-Ubiquitin cleavage activities are affected by OTUB1/2 N-terminal tails. For the quantitation of the relative Ub-cleavage shown in (F), the sum of the intensities of the bands corresponding to cleaved tetra-ubiquitin (Ub/Ub2/Ub3 observed in (G), upper panel) was normalized to the intensity of the band corresponding to the enzyme used (observed in the anti-his immunoblot, (G), bottom panel). All values in (F) are shown relative to the values observed for OTUB2 (mean of OTUB2 is set to 1), and the error bars are S.E. (n = 4).
Figure 7.
Structural features determining OTUB2’s broader cleavage specificity as compared to OTUB1.
The structural models of (A) OTUB1 (adapted from 14) and (B) OTUB2 (this study) are shown in blue, the proximal ubiquitin in purple and the distal ubiquitin in red (note that the proximal ubiquitin (purple) in (B) is not part of the structure). The N-terminal α-helix of OTUB1 (dark blue cylinder) that is absent in OTUB2 makes direct contact with the proximal ubiquitin and hence restricts its binding to an orientation presenting Lys48 towards the catalytic site (red arrows). This restriction is not present in OTUB2, thereby allowing a more permissive ubiquitin recognition mode.