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Intrinsic disorder drives N-terminal ubiquitination by Ube2w

Abstract

Ubiquitination of the αN-terminus of protein substrates has been reported sporadically since the early 1980s. However, the identity of an enzyme responsible for this unique ubiquitin (Ub) modification has only recently been elucidated. We show the Ub-conjugating enzyme (E2) Ube2w uses a unique mechanism to facilitate the specific ubiquitination of the α-amino group of its substrates that involves recognition of backbone atoms of intrinsically disordered N termini. We present the NMR-based solution ensemble of full-length Ube2w that reveals a structural architecture unlike that of any other E2 in which its C terminus is partly disordered and flexible to accommodate variable substrate N termini. Flexibility of the substrate is critical for recognition by Ube2w, and either point mutations in or the removal of the flexible C terminus of Ube2w inhibits substrate binding and modification. Mechanistic insights reported here provide guiding principles for future efforts to define the N-terminal ubiquitome in cells.

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Figure 1: Ube2w has distinct E2 activity.
Figure 2: Ube2w transfers Ub to flexible/disordered N termini.
Figure 3: The Ube2w C terminus is flexible and occupies a noncanonical position.
Figure 4: NMR ensemble of Ube2w reveals a new E2 architecture.
Figure 5: The Ube2w C terminus is required to interact with substrates.
Figure 6: The Ube2w C terminus facilitates α-amino group reactivity.

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Acknowledgements

We acknowledge D. Christensen and C. Eakin for their initial observations on Ube2w and J.N. Pruneda for collecting SAXS data on Ube2w. This work was supported by National Institute of General Medical Sciences grants R01 GM088055 (REK), R01 GM098503 (P.S.B.), K99 NS073936 (K.M.S.) and R01 AG034228 (H.L.P.) and the University of Washington Hurd Fellowship Fund and Public Health Service National Research Service Award 2T32 GM007270 (to V.V.).

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V.V., P.S.B. and R.E.K. conceived the experiments and wrote the manuscript. V.V. performed the biochemical and structural experiments with help from K.M.S. and E.D.D. D.M.W. performed the initial characterization of Ube2w. V.B. and K.S.J.E.-J. performed MS. L.S. and D.B. performed the structure calculations. H.L.P. provided guidance. R.E.K. supervised the project.

Corresponding author

Correspondence to Rachel E Klevit.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–16 and Supplementary Tables 1–3. (PDF 21157 kb)

Supplementary Data Set 1

Raw experimental RDC values for anisotropic and isotropic N-H spectra collected with and without Pf1 phage are provided. (XLSX 53 kb)

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Vittal, V., Shi, L., Wenzel, D. et al. Intrinsic disorder drives N-terminal ubiquitination by Ube2w. Nat Chem Biol 11, 83–89 (2015). https://doi.org/10.1038/nchembio.1700

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