Abstract
During ubiquitin ligation, an E2 conjugating enzyme receives ubiquitin from an E1 enzyme and then interacts with an E3 ligase to modify substrates. Competitive binding experiments with three human E2-E3 protein pairs show that the binding of E1s and of E3s to E2s are mutually exclusive. These results imply that polyubiquitination requires recycling of E2 for addition of successive ubiquitins to substrate.
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Acknowledgements
We thank P. Howley and J. Huibregtse for clones of the E6AP-HECT domain and UbcH7. B.K. acknowledges funding from the US National Institutes of Health (NIH), the Searle Scholar's Program and the Beckman Foundation. B.A.S. acknowledges funding from American Lebanese Syrian Associated Charities (ALSAC), the NIH (P30CA21765, R01GM69530), the Philip and Elizabeth Gross Foundation, the Beckman Foundation and the Pew Scholar's Program. D.M.D. acknowledges support from the American Cancer Society.
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Supplementary information
Supplementary Fig. 1
Enhanced affinity UbcH7–E6AP-HECT pair inhibits E1-mediated UbcH7 transthiolation at lower E6AP-HECT concentrations. (PDF 76 kb)
Supplementary Fig. 2
Kinetics of E1-mediated UbcH7–32P-ubiquitin formation in the presence and absence of high concentrations of E6AP-HECT. (PDF 188 kb)
Supplementary Table 1
Measured dissociation and inhibition constants for wild type and mutant UbcH7-E6AP-HECT interactions. (PDF 28 kb)
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Eletr, Z., Huang, D., Duda, D. et al. E2 conjugating enzymes must disengage from their E1 enzymes before E3-dependent ubiquitin and ubiquitin-like transfer. Nat Struct Mol Biol 12, 933–934 (2005). https://doi.org/10.1038/nsmb984
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DOI: https://doi.org/10.1038/nsmb984
