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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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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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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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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DOI: https://doi.org/10.1038/nsmb.2108
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