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Ubiquitin chain conformation regulates recognition and activity of interacting proteins

Abstract

Mechanisms of protein recognition have been extensively studied for single-domain proteins1, but are less well characterized for dynamic multidomain systems. Ubiquitin chains represent a biologically important multidomain system that requires recognition by structurally diverse ubiquitin-interacting proteins2,3. Ubiquitin chain conformations in isolation are often different from conformations observed in ubiquitin-interacting protein complexes, indicating either great dynamic flexibility or extensive chain remodelling upon binding. Using single-molecule fluorescence resonance energy transfer, we show that Lys 63-, Lys 48- and Met 1-linked diubiquitin exist in several distinct conformational states in solution. Lys 63- and Met 1-linked diubiquitin adopt extended ‘open’ and more compact ‘closed’ conformations, and ubiquitin-binding domains and deubiquitinases (DUBs) select pre-existing conformations. By contrast, Lys 48-linked diubiquitin adopts predominantly compact conformations. DUBs directly recognize existing conformations, but may also remodel ubiquitin chains to hydrolyse the isopeptide bond. Disruption of the Lys 48–diubiquitin interface changes conformational dynamics and affects DUB activity. Hence, conformational equilibria in ubiquitin chains provide an additional layer of regulation in the ubiquitin system, and distinct conformations observed in differently linked polyubiquitin may contribute to the specificity of ubiquitin-interacting proteins.

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Figure 1: Ubiquitin chain conformations and ensemble FRET measurements.
Figure 2: Single-molecule FRET of K48NC (a), K63NC (b) and M1NC (c).
Figure 3: Ubiquitin-interacting protein binding to Lys 63- and Met 1-linked chains.
Figure 4: DUB interaction with Lys48-linked chains.

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Acknowledgements

We would like to thank members of the Komander, Jackson and Klenerman laboratories, R. Williams, S. Freund, C. Johnson, S. McLaughlin and A. Fersht for discussions. Work in the Komander laboratory is supported by the Medical Research Council (U105192732) and the EMBO Young Investigator Program. G.B. and S.I. were supported by the BBSRC, the Newton Trust and an EMBO YIP small grant to D.Ko. Work in the Klenerman laboratory is supported by EPSRC.

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Contributions

Y.Y., G.B. and M.H.H. designed and performed the experiments, including single-molecule measurements, and analysed the data. Y.Y. and G.B. generated all proteins used in this study. Y.Y. performed kinetic experiments. M.H.H. and S.I. built the PAX instrument and A.A.Z. programmed the control for PAX measurements. S.I. performed single molecule experiments and contributed to data analysis. M.J.R.-R. and A.O. performed lifetime measurements. D.Kl., S.E.J. and D.Ko. directed the research and analysed the results. All authors contributed to the writing of the manuscript.

Corresponding authors

Correspondence to David Klenerman, Sophie E. Jackson or David Komander.

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The authors declare no competing financial interests.

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Ye, Y., Blaser, G., Horrocks, M. et al. Ubiquitin chain conformation regulates recognition and activity of interacting proteins. Nature 492, 266–270 (2012). https://doi.org/10.1038/nature11722

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