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The mechanism of OTUB1-mediated inhibition of ubiquitination

Nature volume 483, pages 618–622 (2012)Cite this article

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Abstract

Histones are ubiquitinated in response to DNA double-strand breaks (DSB), promoting recruitment of repair proteins to chromatin1. UBC13 (also known as UBE2N) is a ubiquitin-conjugating enzyme (E2) that heterodimerizes with UEV1A2 (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 thiolester4. Residues amino-terminal to the OTU domain, which had been implicated in ubiquitin binding4, are required for binding to UBC13∼Ub and inhibition of K63Ub synthesis5. 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 transfer6,7. 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 enzymes4 in a non-catalytic manner.

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Figure 1: Allosteric regulation of OTUB1 by ubiquitin.
Figure 2: Structure OTUB1–UBC13 and OTUB1–Ubal–UBC13 DCA ∼Ub.
Figure 3: Conformational changes in the OTU domain triggered by Ubal binding.
Figure 4: OTUB1 N-terminal arm and the mechanism of E2 inhibition.

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Primary accessions

Protein Data Bank

Data deposits

Coordinates and diffraction amplitudes are deposited in the Protein Data Bank under accession numbers 4DHI (worm OTUB1–UBC13), 4DHJ (worm OTUB1–Ubal–UBC13DCA∼Ub) and 4DHZ (human/worm OTUB1–Ubal– UBC13DCA∼Ub).

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Acknowledgements

We thank E. Henderson for generating the human/worm OTUB1 clone and C. Berndsen, A. DiBello, A. Datta and M. Bianchet for discussions. GM/CA-CAT has been funded in whole or in part with funds from the National Cancer Institute (Y1-CO-1020) and the National Institute of General Medical Science (Y1-GM-1104). Use of the Advanced Photon Source was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under contract no. DE-AC02-06CH11357.

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  1. Tao Wang

    Present address: Present address: National Institute of Allergy and Infectious Diseases, Vaccine Research Center, 40 Convent Drive, Bethesda, Maryland 20892, USA,

Authors and Affiliations

  1. Department of Biophysics and Biophysical Chemistry and the Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, 21205, Maryland, USA

    Reuven Wiener, Xiangbin Zhang, Tao Wang & Cynthia Wolberger

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Contributions

R.W. and C.W. designed the experiments and R.W. performed all biochemical experiments. Cloning, expression and protein purification were carried out by X.Z., T.W. and R.W. Complexes were prepared for crystallization and crystals were grown by X.Z. and R.W.; R.W. determined the crystal structure with guidance from C.W.; R.W. and C.W. wrote the manuscript.

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Correspondence to Cynthia Wolberger.

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The authors declare no competing financial interests.

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This file contains Supplementary Figures 1-13 with legends and Supplementary Table 1. (PDF 1423 kb)

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Wiener, R., Zhang, X., Wang, T. et al. The mechanism of OTUB1-mediated inhibition of ubiquitination. Nature 483, 618–622 (2012). https://doi.org/10.1038/nature10911

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