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Essentiality of a non-RING element in priming donor ubiquitin for catalysis by a monomeric E3

Nature Structural & Molecular Biology volume 20, pages 982–986 (2013)Cite this article

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Abstract

RING E3 ligases catalyze the transfer of ubiquitin (Ub) from E2 ubiquitin-conjugating enzyme thioesterified with Ub (E2~Ub) to substrate. For RING E3 dimers, the RING domain of one subunit and tail of the second cooperate to prime Ub, but how this is accomplished by monomeric RING E3s in the absence of a tail-like component is currently unknown. Here, we present a crystal structure of a monomeric RING E3, Tyr363-phosphorylated human CBL-B, bound to a stabilized Ub-linked E2, revealing a similar mechanism in activating E2~Ub. Both pTyr363 and the pTyr363-induced element interact directly with Ub's Ile36 surface, improving the catalytic efficiency of Ub transfer by ~200-fold. Hence, interactions outside the canonical RING domain are crucial for optimizing Ub transfer in both monomeric and dimeric RING E3s. We propose that an additional non-RING Ub-priming element may be a common RING E3 feature.

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Figure 1: Structure of pCBL-B–UbcH5B–Ub–ZAP-70 peptide.
Figure 2: Validation of pTyr363-Ub interactions.
Figure 3: Effect of pTyr363 on the kinetics of Ub transfer.
Figure 4: Requirements for optimal Ub transfer by RING E3s.

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Acknowledgements

We would like to thank A. Schuettelkopf for discussion, W. Clark and A. Keith for in-house DNA sequencing and Diamond Light Source for access to beamlines I04 and I24 beamlines (mx6683) that contributed to the results presented here. This work was supported by Cancer Research UK.

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  1. Hao Dou and Lori Buetow: These authors contributed equally to this work.

Authors and Affiliations

  1. Beatson Institute for Cancer Research, Glasgow, UK

    Hao Dou, Lori Buetow, Gary J Sibbet, Kenneth Cameron & Danny T Huang

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  1. Hao Dou
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Contributions

H.D., L.B. and D.T.H. performed protein purification, crystallization and structure determination. H.D. and L.B. conducted ubiquitination assays. H.D. performed kinetic analyses. K.C. and G.J.S. performed and analyzed NMR experiments. H.D., L.B. and D.T.H. wrote the manuscript.

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Correspondence to Danny T Huang.

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Dou, H., Buetow, L., Sibbet, G. et al. Essentiality of a non-RING element in priming donor ubiquitin for catalysis by a monomeric E3. Nat Struct Mol Biol 20, 982–986 (2013). https://doi.org/10.1038/nsmb.2621

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