Molecular basis of Lys-63-linked polyubiquitination inhibition by the interaction between human deubiquitinating enzyme OTUB1 and ubiquitin-conjugating enzyme UBC13

Abstract

UBC13 is the only known E2 ubiquitin (Ub)-conjugating enzyme that produces Lys-63-linked Ub chain with its cofactor E2 variant UEV1a or MMS2. Lys-63-linked ubiquitination is crucial for recruitment of DNA repair and damage response molecules to sites of DNA double-strand breaks (DSBs). A deubiquitinating enzyme OTUB1 suppresses Lys-63-linked ubiquitination of chromatin surrounding DSBs by binding UBC13 to inhibit its E2 activity independently of the isopeptidase activity. OTUB1 strongly suppresses UBC13-dependent Lys-63-linked tri-Ub production, whereas it allows di-Ub production in vitro. The mechanism of this non-canonical OTUB1-mediated inhibition of ubiquitination remains to be elucidated. Furthermore, the atomic level information of the interaction between human OTUB1 and UBC13 has not been reported. Here, we determined the crystal structure of human OTUB1 in complex with human UBC13 and MMS2 at 3.15 Å resolution. The presented atomic-level interactions were confirmed by surface-plasmon resonance spectroscopy with structure-based mutagenesis. The designed OTUB1 mutants cannot inhibit Lys-63-linked Ub chain formation in vitro and histone ubiquitination and 53BP1 assembly around DSB sites in vivo. Finally, we propose a model for how capping of di-Ub by the OTUB1-UBC13-MMS2/UEV1a complex efficiently inhibits Lys-63-linked tri-Ub formation.

FIGURE 1.

Crystal structure of the OTUB1-ΔN-UBC13-MMS2 ternary complex. a, four UBC13-MMS2 complexes contacting one OTUB1 molecule is shown. OTUB1 is colored orange. Complex A, B, C, and D of the UBC13-MMS2 are colored purple, red, green, and yellow, respectively. b, overall structure of the OTUB1-ΔN-UBC13-MMS2 ternary complex is shown. OTUB1, UBC13, and MMS2 are colored orange, green, and yellow, respectively. c, shown is a stereo view of the interface between OTUB1-ΔN and UBC13.

FIGURE 2.

Phe-133, Phe-138, and Met-211 of OTUB1 are crucial for the inhibition of UBC13-dependent Lys-63 chain formation. In vitro Lys-63 chain synthesis by UBC13-UEV1a was performed in the presence or absence of the indicated recombinant OTUB1. Reaction products were analyzed by Western blotting (IB) for Ub. Reactions after 30 min and 2 h are shown in a and b, respectively.

FIGURE 3.

OTUB1 mutants retain full DUB activities. Lys-48-Ub₄ cleavage by the OTUB1 mutants was analyzed by SDS-PAGE. The reaction products were stained with Ponceau S and immunoblotted (IB) with anti-Ub antibodies.

FIGURE 4.

Phe-133, Phe-138, and Met-211 of OTUB1 are required for the inhibition of UBC13/RNF168-mediated DNA damage response. a, 293T cells cotransfected with hemagglutinin (HA)-RNF168, MYC-ubiquitin, and/or FLAG-OTUB1 were subjected to FLAG immunoprecipitation (IP) or acid extraction of chromatin. WCL, whole cell lysate. b and c, U2OS cells transfected with the indicated FLAG-OTUB1 expression vectors were irradiated with 2 gray (Gy) and processed for 53BP1 and FLAG immunofluorescence 1 h post-ionizing radiation. Scale bar, 25 μm. c, shown is quantification of the data shown in b (mean ± S.D., n = 3). Gy, gray; WCL, whole cell lysate; IP, immunoprecipitation.

FIGURE 5.

OTUB1 mildly inhibits UBC13-dependent di-Ub formation. In vitro Lys-63 chain synthesis by UBC13-UEV1a was performed in the presence of C91S or F133A/F138A/M211A/C91S OTUB1. Reaction products were analyzed by Western blotting for Ub after incubation for the indicated time at 37 °C.

FIGURE 6.

Capping and translocation inhibitions of Lys-63 chain synthesis by OTUB1. Drawing schemes are the same as in Fig. 1. Ub are colored cyan, pink, and red, respectively.