OTUB1 co-opts Lys48-linked ubiquitin recognition to suppress E2 enzyme function

Abstract

Ubiquitylation entails the concerted action of E1, E2, and E3 enzymes. We recently reported that OTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidase activity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme for RNF168. OTUB1 also inhibits E2s of the UBE2D and UBE2E families. Here we elucidate the structural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer. OTUB1 recognizes ubiquitin-charged E2s through contacts with both donor ubiquitin and the E2 enzyme. Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1 to promote formation of the inhibited E2 complex. Lys48 of donor ubiquitin lies near the OTUB1 catalytic site and the C terminus of free ubiquitin, a configuration that mimics the products of Lys48-linked ubiquitin chain cleavage. OTUB1 therefore co-opts Lys48-linked ubiquitin chain recognition to suppress ubiquitin conjugation and the DNA damage response.

Figure 1. A genetic system to dissect E2 inhibition by OTUB1 (see also Figure S1)

(A) Left panel: Five-fold serial dilutions of BY4741 strains transformed with the indicated pRS425 derivatives were spotted onto selective media containing either glucose (uninduced condition) or galactose (induced) as the carbon source. Right panel: Whole cell extracts (WCE) of the strains shown in the left panel were separated by SDS-PAGE and probed for Flag-OTUB1 expression. Pgk1 immunoblotting was used as a loading control. (B) Left panel: Examination of yeast growth assay as in (A). Right panel: WCE of the strains shown in the left panel were separated by SDS-PAGE and probed for Flag-OTUB1 expression. Pgk1 immunoblotting was used as a loading control. (C) Schematic of the genetic screen used to identify human OTUB1 mutants that fail to inhibit yeast growth. (D) Examination of yeast growth as in (A). (E) Whole cell extracts of the strains shown in (D) were separated by SDS-PAGE and probed for Flag-OTUB1 expression. Pgk1 immunoblotting was used as a loading control.

Figure 2. Regulation of RNF168-dependent ubiquitylation by OTUB1 (see also Figure S2)

(A–C) U2OS cells were first transfected with non-targeting (CTRL) or OTUB1 siRNAs and then transfected with the indicated pCDNA5-Flag derivatives. All OTUB1 plasmids were siRNA-resistant. Cells were then irradiated with X-rays (3 Gy) and processed for Flag or conjugated Ub (FK2) immunofluorescence 0, 4 and 24 h post-IR. DNA was counterstained with DAPI. Quantitation of the immunofluorescence data is shown in (A). Data is presented at the mean +/− S.D. n=3. The dashed line indicates the percentage of control-transfected cells with FK2 foci 24 h post-irradiation. Representative micrographs of immunofluorescence data 24 h post-irradiation are shown in (B). Scale bar=10 μm. Analysis of Flag-OTUB1 expression is shown in (C). Tubulin was used a loading control.

Figure 3. Structure of the Ub~UbcH5b-OTUB1-Ub complex (see also Figures S1, S3, S4, S5)

(A) Schematic and ribbons representation of the Ub~UbcH5b-OTUB1-Ub complex with subunits coloured pink, cyan, green, and yellow, respectively. The side chains of the catalytic cysteine of OTUB1 and UbcH5 are shown as sticks. Helix αA of OTUB1 and the N- and C-termini of subunits are labeled where visible. (B) Stereo view of the OTUB1-UbcH5b binding interface. Main chains are coloured as in (A). Contact residues are shown as sticks with carbon atoms coloured according to their respective main chain and oxygen, nitrogen, and sulfur atoms are coloured red, blue, and yellow, respectively. The positions of OTUB1 mutants tested in this study are labeled red. (C) UbcH5c and UBC13 utilize similar surfaces to bind OTUB1. Significant perturbations (>1 S.D. from the mean) arising from OTUB1 binding to ¹⁵N-UbcH5c (top) or ¹⁵N-UBC13 (bottom) are mapped in red onto the respective E2 structures. (D) NMR analysis shows binding of OTUB1 to UbcH5c in solution is consistent with X-ray data. Resonances in ¹⁵N-UbcH5c significantly affected by binding of OTUB1 are mapped (shown in red) onto the corresponding residues in the OTUB1:UbcH5b~Ub crystal structure.

Figure 4. Binding mode of donor Ub to OTUB1 (see also Figure S1 and S3)

(A) Stereo view of the donor Ub-OTUB1 binding interface. Colouring as in Figure 3B. The positions of OTUB1 mutants tested in this study are labeled in red. (B) Stereo comparison of Ub-UIM and donor Ub-OTUB1 helix αA binding modes. The UIM-Ub structures corresponding to VPS27 (PDBID 1Q0W) and Hrs (PDBID 2D3G) are coloured grey and orange respectively. The OTUB1-donor Ub structure is coloured as in Figure 3B. In all three complexes, Ub engages its target α-helices using a common surface centered on Ile44 (labeled). The N- and C-termini of the UIMs and OTUB1 α-helices are also labeled. (C) A mixture of uncharged and Ub-charged UBC13^C87S that was either prepared with wild type Ub (UBC13~Ub) or UbI44A (UBC13~Ub^I44A) were incubated with either GST-OTUB1^C91S or, as control, GST-Pcc1. Proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue.

Figure 5. Binding mode of free Ub to OTUB1 (see also Figure S1, S3 and S6)

(A) Stereo view of the free Ub-OTUB1 binding interface. Coloring is as in Figure 3B. The positions of OTUB1 mutants tested in this study are labeled in red. The side chain of Phe190 in OTUB1, immediately adjacent to Phe189 and Phe193, is not shown for sake of clarity. For the Phe190 position see Figure S5B. (B) Stereo view of donor Ub and free Ub in the proximity of the catalytic cleft of OTUB1. Coloring is as in Figure 3B. Highlighted as sticks are the catalytic residues of OTUB1 (Cys91, His265 and Pro87), and residues in direct contact between donor and free Ub. (C) Stereo view of donor Ub and free Ub in proximity of the catalytic cleft of OTUB1. Coloring is as in Figure 3B. Highlighted as sticks are the catalytic residues of OTUB1 (Cys91, His265 and Pro87), K48 of donor Ub and Gly76 of free Ub. Superimposed on the solved structure is a model of an isopeptide linkage between the K48 side chain of donor Ub and the Gly76 C-terminus of free Ub. A productive conformation of OTUB1 catalytic residues in the Ub~UbcH5b-OTUB1-Ub complex contrasts with a non-productive conformation observed in the apo-OTUB1 complex (PDB 2ZFY).

Figure 6. The OTUB1 mutants map to functionally important intermolecular interfaces

(A) Position of the OTUB1 mutations identified in the yeast genetic screen on the structure-based model of the Ub~UBC13-OTUB1-Ub complex. (B) Single-turnover ubiquitylation reactions were assembled by pre-incubating UBC13, E1, biotinylated Ub and ATP for 5 min with or without the indicated OTUB1 proteins. To the reaction mixtures were added excess unlabeled Ub, UEV1 and EDTA. The reactions were incubated for an additional 15 min, separated by SDS-PAGE, transferred onto nitrocellulose and stained with Ponceau S followed by streptavidin (SA)-HRP. Asterisk denotes a DTT-resistant ubiquitylated UBC13 species. (C) Examination of yeast growth and protein expression as in Figure 1A. (D–E) Graph of internally quenched K48-linked di-Ub cleavage reactions with the indicated OTUB1 proteins.

Figure 7. Potential modulation of the OTUB1-E2 interaction

(A) Examination of yeast growth as in Figure 1A. (B) Left panel: U2OS cells were first transfected with non-targeting (CTRL) or OTUB1 siRNAs and then transfected with the indicated pCDNA3-Flag derivatives and processed as in Figure 2. Quantitation of the immunofluorescence data is shown. Data is presented at the mean +/− S.D. n=3. Right panel: examination of Flag-OTUB1 expression. Tubulin was used as loading control (C) Analysis of di-Ub synthesis by UBC13 in the absence (−) or presence of the indicated OTUB1 proteins exactly as Figure 6B. (D) Titration analysis of OTUB1 binding to UbcH5b~Ub in the absence or presence of 1 or 10 μM free Ub using a time TR-FRET assay. The data presented as the mean +/− S.E.M. n=2. (E) Pull down analysis of GST-OTUB1 binding to free Ub in the presence of 0, 5, 10 and 20 μM concentrations of UbcH5b~Ub. Proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue.