Article | Published:

Molecular architecture and mechanism of the anaphase-promoting complex

Nature volume 513, pages 388393 (18 September 2014) | Download Citation

Abstract

The ubiquitination of cell cycle regulatory proteins by the anaphase-promoting complex/cyclosome (APC/C) controls sister chromatid segregation, cytokinesis and the establishment of the G1 phase of the cell cycle. The APC/C is an unusually large multimeric cullin-RING ligase. Its activity is strictly dependent on regulatory coactivator subunits that promote APC/C–substrate interactions and stimulate its catalytic reaction. Because the structures of many APC/C subunits and their organization within the assembly are unknown, the molecular basis for these processes is poorly understood. Here, from a cryo-electron microscopy reconstruction of a human APC/C–coactivator–substrate complex at 7.4 Å resolution, we have determined the complete secondary structural architecture of the complex. With this information we identified protein folds for structurally uncharacterized subunits, and the definitive location of all 20 APC/C subunits within the 1.2 MDa assembly. Comparison with apo APC/C shows that the coactivator promotes a profound allosteric transition involving displacement of the cullin-RING catalytic subunits relative to the degron-recognition module of coactivator and APC10. This transition is accompanied by increased flexibility of the cullin-RING subunits and enhanced affinity for UBCH10–ubiquitin, changes which may contribute to coactivator-mediated stimulation of APC/C E3 ligase activity.

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Accessions

Electron Microscopy Data Bank

Data deposits

Electron microscopy maps are deposited with the EMDataBank with accession codes: EMD-2651 (ternary), EMD-2652 (apo), EMD-2653 (APC/C–APC11ΔRING) and EMD-2654 (APC/CΔAPC15).

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Acknowledgements

We thank P. da Fonseca for her guidance preparing cryo electron microscopy grids and X. Bai and S. Scheres for help with the use of RELION and A. Plechanovova for advice in preparing stable UBCH10–Ub conjugates. We thank D. Morgan and W. Chao for their comments on the manuscript and D. Morgan for communicating data before publication. This work was funded by a Cancer Research UK grant to D.B.

Author information

Author notes

    • Leifu Chang
    • , Ziguo Zhang
    • , Jing Yang
    •  & David Barford

    Present address: MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK (L.C., Z.Z., J.Y. and D.B.).

    • Leifu Chang
    •  & Ziguo Zhang

    These authors contributed equally to this work.

Affiliations

  1. Division of Structural Biology, Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK

    • Leifu Chang
    • , Ziguo Zhang
    • , Jing Yang
    •  & David Barford
  2. MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK

    • Leifu Chang
    • , Ziguo Zhang
    • , Jing Yang
    • , Stephen H. McLaughlin
    •  & David Barford

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Contributions

L.C. prepared grids, collected and analysed electron microscopy data and determined the three dimensional reconstructions, fitted coordinates and built models, prepared figures and co-wrote the paper. Z.Z. designed and made constructs, performed biochemical analysis and purified proteins. J.Y. prepared and purified the complexes. S.H.McL. performed and analysed SPR experiments. D.B. directed the project, built models and co-wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to David Barford.

Extended data

Supplementary information

Videos

  1. 1.

    Assembly and architecture of human APC/C

    Video shows the structure of the human APC/CCdh1.Hsl1 ternary complex at 7.4 Å resolution. The molecular envelope colour-coded according to subunit assignments is shown. Second, the video shows the fitting of TPR subunit Apc6 and TPR accessory subunit Apc12 to EM density. The homo-dimers of Apc8, Apc6, Apc3 and Apc7 stack in parallel to form a left-handed supra-helix that maximises protein interfaces. Third, the video focuses on the platform subunits, Apc5, Apc15, Apc4, the Apc2-Apc11 catalytic module and Apc1. Fourth the degron recognition subunits Apc10 and Cdh1 together with EM density for the D box and KEN box are shown.

  2. 2.

    Cdh1-mediates conformational change of the APC/C

    Video morphs between apo APC/C and the APC/CCdh1.Hsl1 ternary complex, showing how the Cdh1 coactivator induces conformational changes of the platform subunits Apc2-Apc11, Apc4 and Apc5. The video shows morphing between the molecular envelopes followed by morphing between atomic models of the complex. Structures were first aligned using all subunits. Later, superimposing using the Apc1 PC domain as a reference indicates that the conformational change involves a rotation of the platform about an axis centred close to the Apc1PC-Apc8 interface. The insertion of Cdh1NTD at this interface disrupts Apc1PC-Apc8 interactions, shifting the platform subunits, displacing the Apc2CTD-Apc11 catalytic module.

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DOI

https://doi.org/10.1038/nature13543

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