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Mechanism of ubiquitylation by dimeric RING ligase RNF4

Nature Structural & Molecular Biology volume 18, pages 1052–1059 (2011)Cite this article

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Abstract

Mammalian RNF4 is a dimeric RING ubiquitin E3 ligase that ubiquitylates poly-SUMOylated proteins. We found that RNF4 bound ubiquitin-charged UbcH5a tightly but free UbcH5a weakly. To provide insight into the mechanism of RING-mediated ubiquitylation, we docked the UbcH5~ubiquitin thioester onto the RNF4 RING structure. This revealed that with E2 bound to one monomer of RNF4, the thioester-linked ubiquitin could reach across the dimer to engage the other monomer. In this model, the 'Ile44 hydrophobic patch' of ubiquitin is predicted to engage a conserved tyrosine located at the dimer interface of the RING, and mutation of these residues blocked ubiquitylation activity. Thus, dimeric RING ligases are not simply inert scaffolds that bring substrate and E2-loaded ubiquitin into close proximity. Instead, they facilitate ubiquitin transfer by preferentially binding the E2~ubiquitin thioester across the dimer and activating the thioester bond for catalysis.

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Figure 1: Structure of dimeric RING domain of RNF4.
Figure 2: Ubiquitylation by RNF4 can proceed both in cis and in trans.
Figure 3: RNF4 preferentially binds ubiquitin-loaded E2 and activates the bond between E2 and ubiquitin.
Figure 4: The Ile44-centered hydrophobic patch on ubiquitin is required for RNF4-mediated ubiquitylation.
Figure 5: Tyr193, a residue located at the dimer interface of the RNF4 RING domain, is required for activation of the thioester bond in the E2~ubiquitin thioester.
Figure 6: A linear fusion of full-length RNF4 and the RNF4 RING domain shows that E2-binding site in one RING domain and Tyr193 in the other RING are both required for ubiquitylation activity.

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Acknowledgements

We thank C. Botting for mass spectrometric analysis, M. Agacan for analytical ultracentrifugation analysis and N. Wood for help with cloning. His-Ube1 was a kind gift from the Division of Signal Transduction Therapy, University of Dundee. The sequence encoding RNF4 ΔC in pLou3 vector was a kind gift from L. Shen, University of Dundee. A.P. was funded by a studentship from the Wellcome Trust. This work was supported by a grant to R.T.H. from Cancer Research UK. The structural biology was supported by the Scottish Funding Council (reference SULSA) and by the UK Biotechnology and Biological Sciences Research Council through the SPoRT initiative.

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Authors and Affiliations

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

    Anna Plechanovová, Ellis G Jaffray & Ronald T Hay

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

    Stephen A McMahon, Kenneth A Johnson & James H Naismith

  3. Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, UK

    Iva Navrátilová

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Contributions

A.P. purified RNF4 proteins, carried out crystallography, conducted biochemical analysis and interpreted the data. E.G.J. purified recombinant proteins and carried out ubiquitylation assays. S.A.M., K.A.J. and J.H.N. contributed to structural analysis. I.N. contributed to biochemical analysis. A.P., J.H.N. and R.T.H. wrote the paper. R.T.H. conceived the project and contributed to data interpretation.

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Correspondence to Ronald T Hay.

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Plechanovová, A., Jaffray, E., McMahon, S. et al. Mechanism of ubiquitylation by dimeric RING ligase RNF4. Nat Struct Mol Biol 18, 1052–1059 (2011). https://doi.org/10.1038/nsmb.2108

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