The mechanism of OTUB1-mediated inhibition of ubiquitination

Abstract

Histones are ubiquitinated in response to DNA double-strand breaks (DSB), promoting recruitment of repair proteins to chromatin. UBC13 (also known as UBE2N) is a ubiquitin-conjugating enzyme (E2) that heterodimerizes with UEV1A (also known as UBE2V1) and synthesizes K63-linked polyubiquitin (K63Ub) chains at DSB sites in concert with the ubiquitin ligase (E3), RNF168 (ref. 3). K63Ub synthesis is regulated in a non-canonical manner by the deubiquitinating enzyme, OTUB1 (OTU domain-containing ubiquitin aldehyde-binding protein 1), which binds preferentially to the UBC13∼Ub thiolester. Residues amino-terminal to the OTU domain, which had been implicated in ubiquitin binding, are required for binding to UBC13∼Ub and inhibition of K63Ub synthesis. Here we describe structural and biochemical studies elucidating how OTUB1 inhibits UBC13 and other E2 enzymes. We unexpectedly find that OTUB1 binding to UBC13∼Ub is allosterically regulated by free ubiquitin, which binds to a second site in OTUB1 and increases its affinity for UBC13∼Ub, while at the same time disrupting interactions with UEV1A in a manner that depends on the OTUB1 N terminus. Crystal structures of an OTUB1-UBC13 complex and of OTUB1 bound to ubiquitin aldehyde and a chemical UBC13∼Ub conjugate show that binding of free ubiquitin to OTUB1 triggers conformational changes in the OTU domain and formation of a ubiquitin-binding helix in the N terminus, thus promoting binding of the conjugated donor ubiquitin in UBC13∼Ub to OTUB1. The donor ubiquitin thus cannot interact with the E2 enzyme, which has been shown to be important for ubiquitin transfer. The N-terminal helix of OTUB1 is positioned to interfere with UEV1A binding to UBC13, as well as with attack on the thiolester by an acceptor ubiquitin, thereby inhibiting K63Ub synthesis. OTUB1 binding also occludes the RING E3 binding site on UBC13, thus providing a further component of inhibition. The general features of the inhibition mechanism explain how OTUB1 inhibits other E2 enzymes in a non-catalytic manner.

Figure 1. Allosteric regulation of OTUB1 by ubiquitin

A. Schematic diagram of OTUB1 illustrating proximal and distal ubiquitin binding sites. B. Effect of ubiquitin aldehyde (Ubal) on the ability of hOTUB1 to inhibit K63 polyubiquitin synthesis by UBC13/UEV1a. Assays include 0.1 µM E1, 0.4 µM UBC13/UEV1a, 0.5 µM hOTUB1, 5 µM ubiquitin. The 3 hr time point is shown in the presence (right) and absence (left) of hOTUB1, without (−) and with (+) 0.5 µM Ubal. Top shows detection by anti-Ub Western blot; Coomassie staining below shows level of hOTUB1. C. Pull down assay showing binding of H₆-tagged hOTUB1 to a mixture of UBC13 and UBC13~Ub oxyester in the presence and absence 100 µM free ubiquitin (WT or mutant). D. Effect of hOTUB1 distal site mutations on inhibition of K63Ub synthesis. Assay performed as in (B) but with 1 µM OTUB1, showing 0.5 and 1 hr time points. E. Effect of hOTUB1 N-terminal deletions of 15, 30, 37, and 42 residues on inhibition of K63Ub synthesis by UBC13/UEV1a. Assay performed as in (D). F. Gel filtration showing complex formation between fluorescein-labelled UEV1a (UEV1a*), UBC13 and hOTUB1. Signal due to UEV1a only was monitored at 495 nm. G. Experiment performed as in (F) showing complex formation of UEV1a*, UBC13^DCA~Ub^G75C and hOTUB1 in the absence (red) and presence (green) of Ubal. H. Experiment performed as in (F) but with hOTUB1Δ37. The position at which free UEV1* migrates is indicated. I. Experiment performed as in (F) with fluorescein-labeled UEV mixed with UBC13^DCA~Ub^G75C and OTUB1 samples prepared in the presence and absence of 200 µM ubiquitin. The position at which free UEV1* migrates is indicated.

Figure 2. Structure OTUB1-UBC13 and OTUB1-Ubal-UBC13^DCA~Ub

A. Complex of ceOTUB1 (green) bound to human UBC13 (blue). Respective active site cysteines are shown as space-filling representations. Dashed line indicates disordered residues. B. Contacts at ceOTUB1 (green) -UBC13 (blue) interface. C. Superposition of UBCH5b (UBE2D2, PDB ID 2ESK) and UBCH7 (UBE2L3, PDB ID 1FBV) with UBC13 in the complex with ceOTUB1. UBCH7 contains an insertion (at N94) and a lysine (K96) that would interfere with binding. D. Structure of hybrid h/ceOTUB1 (green) bound to Ubal (distal Ub, yellow), UBC13 (blue) and ubiquitin (proximal Ub, red) that is covalently linked to the active site cysteine (C87) of UBC13 by a DCA linkage. Dashed line indicated disordered C-terminal residues 73–76 of the donor ubiquitin and DCA linkage. E. A 90° rotation compared to (D) showing positions of ceOTUB1 and UBC13 active site cysteine and modeled location of K48 of the proximal ubiquitin. F. Contacts between the donor ubiquitin (red) and the OTU domain (green) in the ceOTUB1-Ubal-UBC13^DCA~Ub complex.

Figure 3. Conformational changes in the OTU domain triggered by Ubal binding

A. Superposition of ceOTUB1 (green) bound to Ubal (yellow surface) with the structure of apo ceOTUB1(gray). Dotted circles indicate regions of conformational change, which are illustrated in the figure panels noted. B. Location of hOTUB1 distal site mutations that affect inhibition. The structure of hOTUB1 (2ZFY; brown) is superimposed on ceOTUB1 (green) – Ubal (yellow). Ubiquitin residues L8 and I44, where substitutions with alanine disrupt allosteric effect of ubiquitin binding, are shown. View is 180° rotation about vertical compared with (A). C. Structural differences in the OTU domain in the presence (green) and absence (gray) of distal Ub that affect contacts with the donor Ub. Side chain conformations for ceOTUB1 in presence and absence of Ubal are shown for R236 and Y233. Dotted lines indicate hydrogen bonds and salt bridges. View shown is from “top” of complex as shown on right of panel (A), rotated 90° counterclockwise. D. Effect of mutating OTUB1 conserved arginine, ceOTUB1-R236E and hOTUB1-R238E, on inhibition of UBC13/UEV1a. Assay performed as in panel 1A, with 1 µM hOTUB1 and 15 µM ceOTUB1. E. View of OTUB domain structural rearrangements colored as in (C). View as in panel (A); proximal ubiquitin not shown. F. Detailed view of catalytic triad in the presence and absence of Ubal (carbon colored as in (C).

Figure 4. OTUB1 N-terminal arm and the mechanism of E2 inhibition

A. Sequence alignment of N-terminal arms of hOTUB1 (top) and ceOTUB1 (bottom). Boxed residues form a helix in the quaternary complex structures containing Ubal and UBC13^DCA~Ub; additional shaded residues in ceOTUB1 are ordered in complex 1 but are not helical. B. Donor Ub (red) interactions with the ceOTUB1 N-terminal helix (green); Ubc13 shown in blue. Dashed lines indicate disordered residues. C. Interactions with the hOTUB1 N-terminal helix of the h/ceOTUB1 hybrid, depicted as in (B). D. Superposition comparing RAP80 (3A1Q) binding to ubiquitin with hOTUB1 N-terminal helix. Two views are shown. E. Superposition of h/ceOTUB1-Ubal-UBC13^DCA~Ub with UBC13/UEV1 (1J7D) showing predicted position of UEV1 (gray). The solvent-accessible surface of the human N-terminal arm residues of OTUB1 is depicted. F. Superposition with quaternary complex showing relative position of the TRAF6 E3 ligase (3HCT).