Abstract
Ubiquitin modification is mediated by a large family of specificity determining ubiquitin E3 ligases. To facilitate ubiquitin transfer, RING E3 ligases bind both substrate and a ubiquitin E2 conjugating enzyme linked to ubiquitin via a thioester bond, but the mechanism of transfer has remained elusive. Here we report the crystal structure of the dimeric RING domain of rat RNF4 in complex with E2 (UbcH5A) linked by an isopeptide bond to ubiquitin. While the E2 contacts a single protomer of the RING, ubiquitin is folded back onto the E2 by contacts from both RING protomers. The carboxy-terminal tail of ubiquitin is locked into an active site groove on the E2 by an intricate network of interactions, resulting in changes at the E2 active site. This arrangement is primed for catalysis as it can deprotonate the incoming substrate lysine residue and stabilize the consequent tetrahedral transition-state intermediate.
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Acknowledgements
We thank M. Alphey and E. Branigan for assistance with data collection. His–UBE1 was a gift from the Division of Signal Transduction Therapy, University of Dundee. CHIP was a gift from A. Knebel and P. Cohen. A.P. was funded by the Wellcome Trust. This work was supported by a grant to R.T.H. from Cancer Research UK. Structural biology was supported by Scottish Funding Council (ref SULSA) and Wellcome Trust (program grant JHN).
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A.P. cloned, expressed and purified proteins, carried out structural analysis, conducted biochemical experiments and interpreted the data. E.G.J. purified recombinant proteins and carried out biochemical analysis. M.H.T. carried out mass spectrometry analysis. J.H.N. contributed to structural analysis and data analysis. A.P., J.H.N. and R.T.H. wrote the paper. R.T.H. conceived the project and contributed to data analysis.
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Plechanovová, A., Jaffray, E., Tatham, M. et al. Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis. Nature 489, 115–120 (2012). https://doi.org/10.1038/nature11376
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DOI: https://doi.org/10.1038/nature11376
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