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BIRC7–E2 ubiquitin conjugate structure reveals the mechanism of ubiquitin transfer by a RING dimer

Abstract

Certain RING ubiquitin ligases (E3s) dimerize to facilitate ubiquitin (Ub) transfer from ubiquitin-conjugating enzyme (E2) to substrate, but structural evidence on how this process promotes Ub transfer is lacking. Here we report the structure of the human dimeric RING domain from BIRC7 in complex with the E2 UbcH5B covalently linked to Ub (UbcH5BUb). The structure reveals extensive noncovalent donor Ub interactions with UbcH5B and both subunits of the RING domain dimer that stabilize the globular body and C-terminal tail of Ub. Mutations that disrupt these noncovalent interactions or RING dimerization reduce UbcH5BUb binding affinity and ubiquitination activity. Moreover, NMR analyses demonstrate that BIRC7 binding to UbcH5BUb induces peak-shift perturbations in the donor Ub consistent with the crystallographically-observed Ub interactions. Our results provide structural insights into how dimeric RING E3s recruit E2Ub and optimize the donor Ub configuration for transfer.

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Figure 1: Structure of BIRC7239–C–UbcH5BRASUb.
Figure 2: UbcH5B–Ub interactions within the complex.
Figure 3: Donor Ub–RING interactions are required for BIRC7-catalyzed reactions.
Figure 4: Ub interaction surface in BIRC7239–C–UbcH5BRASUb, identified by NMR.
Figure 5: Effect of donor Ub interactions on the kinetics of Ub transfer.

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Acknowledgements

We would like to thank K. Vousden and A. Schuettelkopf for discussion. We thank A. Krishnan, a former lab technician, for purification of BIRC3541–C; W. Clark and A. Keith for in-house DNA sequencing; and the Diamond Light Source (DLS) for access to beamlines I04 and I24 beamlines (mx6683) that contributed to the results presented here. This work was funded by Cancer Research UK.

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H.D., L.B. and D.T.H. performed protein purification, assembly of complexes, crystallization and structure determination. H.D. and L.B. conducted and analyzed ubiquitination assays. G.J.S. performed and analyzed SPR experiments. K.C. and G.J.S. performed and analyzed NMR experiments. H.D., L.B. and D.T.H. wrote the manuscript.

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Correspondence to Danny T Huang.

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Dou, H., Buetow, L., Sibbet, G. et al. BIRC7–E2 ubiquitin conjugate structure reveals the mechanism of ubiquitin transfer by a RING dimer. Nat Struct Mol Biol 19, 876–883 (2012). https://doi.org/10.1038/nsmb.2379

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