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Molecular architecture and assembly of the DDB1–CUL4A ubiquitin ligase machinery

Nature volume 443, pages 590–593 (2006)Cite this article

Abstract

Protein ubiquitination is a common form of post-translational modification that regulates a broad spectrum of protein substrates in diverse cellular pathways1. Through a three-enzyme (E1–E2–E3) cascade, the attachment of ubiquitin to proteins is catalysed by the E3 ubiquitin ligase, which is best represented by the superfamily of the cullin-RING complexes2,3. Conserved from yeast to human, the DDB1–CUL4–ROC1 complex is a recently identified cullin-RING ubiquitin ligase, which regulates DNA repair4,5,6,7,8,9,10, DNA replication11,12,13,14 and transcription15, and can also be subverted by pathogenic viruses to benefit viral infection16. Lacking a canonical SKP1-like cullin adaptor and a defined substrate recruitment module, how the DDB1–CUL4–ROC1 E3 apparatus is assembled for ubiquitinating various substrates remains unclear. Here we present crystallographic analyses of the virally hijacked form of the human DDB1–CUL4A–ROC1 machinery, which show that DDB1 uses one β-propeller domain for cullin scaffold binding and a variably attached separate double-β-propeller fold for substrate presentation. Through tandem-affinity purification of human DDB1 and CUL4A complexes followed by mass spectrometry analysis, we then identify a novel family of WD40-repeat proteins, which directly bind to the double-propeller fold of DDB1 and serve as the substrate-recruiting module of the E3. Together, our structural and proteomic results reveal the structural mechanisms and molecular logic underlying the assembly and versatility of a new family of cullin-RING E3 complexes.

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Figure 1: Crystal structure of the DDB1–CUL4A–ROC1 ubiquitin ligase complex hijacked by the V protein of simian virus 5.
Figure 2: Binding interfaces and structural flexibility of DDB1 on CUL4A.
Figure 3: Identification of a novel family of WD40 proteins as the potential substrate receptors for the DDB1–CUL4A E3 machinery.

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Acknowledgements

We thank the beamline staff of the Advanced Light Source at Berkeley for help with data collection, P. Zhou, W. Xu, J. Beavo, Z. Gao and members of the Zheng and Moon laboratories for discussions and help. We also thank J. W. Harper and Y. Xiong for communicating unpublished results. This work is supported by grants from the National Institutes of Health (to N.Z. and M.J.M.), the Pew Scholar Program (N.Z.), and the Howard Hughes Medical Institute (S.A. and R.T.M.). Author Contributions S.A. and T.L. contributed equally to this work.

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Author notes

  1. Stephane Angers & Ti Li

    Present address: Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, M5S 3M2, Canada

  2. Stephane Angers: *These authors contributed equally to this work

Authors and Affiliations

  1. Howard Hughes Medical Institute,

    Stephane Angers & Randall T. Moon

  2. Department of Pharmacology,

    Stephane Angers, Ti Li, Xianhua Yi, Randall T. Moon & Ning Zheng

  3. Department of Genome Sciences,

    Michael J. MacCoss

  4. Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Box 357280, Washington, 98195, Seattle, USA

    Randall T. Moon

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Correspondence to Randall T. Moon or Ning Zheng.

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Competing interests

Structure coordinates for the DDB1–CUL4A–ROC1–SV5-V complex have been deposited in the Protein Data Bank under accession number 2HYE. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Notes

This file contains Supplementary Tables 1–3 showing detailed crystallographic and proteomic results, and Supplementary Figures 1–5 describing more structural and co-affinity interaction analyses. (PDF 1250 kb)

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Angers, S., Li, T., Yi, X. et al. Molecular architecture and assembly of the DDB1–CUL4A ubiquitin ligase machinery. Nature 443, 590–593 (2006). https://doi.org/10.1038/nature05175

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