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E2 ubiquitin-conjugating enzymes regulate the deubiquitinating activity of OTUB1


OTUB1 is a Lys48-specific deubiquitinating enzyme that forms a complex in vivo with E2 ubiquitin (Ub)-conjugating enzymes including UBC13 and UBCH5. OTUB1 binds E2~Ub thioester intermediates and prevents ubiquitin transfer, thereby noncatalytically inhibiting accumulation of polyubiquitin. We report here that a second role of OTUB1-E2 interactions is to stimulate OTUB1 cleavage of Lys48 polyubiquitin. This stimulation is regulated by the ratio of charged to uncharged E2 and by the concentration of Lys48-linked polyubiquitin and free ubiquitin. Structural and biochemical studies of human and worm OTUB1 and UBCH5B show that the E2 enzyme stimulates binding of the Lys48 polyubiquitin substrate by stabilizing folding of the OTUB1 N-terminal ubiquitin-binding helix. Our results suggest that OTUB1–E2 complexes in the cell are poised to regulate polyubiquitin chain elongation or degradation in response to changing levels of E2 charging and available free ubiquitin.

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Figure 1: E2 enzymes stimulate OTUB1 DUB activity.
Figure 2: OTUB1-E2 interactions are required for stimulation of DUB activity.
Figure 3: UBCH5B stabilizes the OTUB1 ubiquitin-binding helix.
Figure 4: Effect of free ubiquitin and E2 charging on OTUB1 DUB activity.
Figure 5: E2 enzymes exist in both charged and uncharged states in vivo.

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We thank S. Urbé and J.W. Harper for advice and suggestions. This work was supported in part by US National Institutes of Health grant GM-060980 (M.J.M.) and National Science Foundation grant MCB-0920082 (C.W.).

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R.W., A.T.D. and C.W. designed experiments, interpreted data and wrote the paper. R.W. and A.T.D. carried out the enzymatic assays and binding studies, and A.T.D. determined the crystal structure and did western blot assays. X.Z. purified proteins and prepared and crystallized the OTUB1–Ubal–UBCH5B~Ub complex. P.M.L. assayed E2–OTUB1 activity on different polyubiquitin chains. C.M.G. performed cell-based experiments under the guidance of M.J.M.

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Correspondence to Cynthia Wolberger.

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The authors declare no competing financial interests.

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Wiener, R., DiBello, A., Lombardi, P. et al. E2 ubiquitin-conjugating enzymes regulate the deubiquitinating activity of OTUB1. Nat Struct Mol Biol 20, 1033–1039 (2013).

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