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Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis

Nature volume 489, pages 115–120 (2012)Cite this article

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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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Figure 1: Structure of the RNF4 RING bound to ubiquitin-loaded UbcH5A.
Figure 2: Molecular interfaces in the RNF4 RING–UbcH5A–Ub complex.
Figure 3: Mutational analysis of the RNF4 RING–UbcH5A–Ub complex.
Figure 4: The same interfaces in E2 and ubiquitin are important for CHIP and RNF4 activity.
Figure 5: E3-mediated structural changes associated with the catalytically primed form of UbcH5A–Ub.

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Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors of the RNF4 RING– UbcH5A(S22R/C85K)–Ub complex were deposited in the Protein Data Bank under accession code 4AP4.

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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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Authors and Affiliations

  1. Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK,

    Anna Plechanovová, Ellis G. Jaffray, Michael H. Tatham & Ronald T. Hay

  2. Biomedical Sciences Research Complex, University of St Andrews, St Andrews KY16 9ST, UK,

    James H. Naismith

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  1. Anna Plechanovová
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Contributions

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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Correspondence to Ronald T. Hay.

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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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