Structures of APC/CCdh1 with substrates identify Cdh1 and Apc10 as the D-box co-receptor

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Abstract

The ubiquitylation of cell-cycle regulatory proteins by the large multimeric anaphase-promoting complex (APC/C) controls sister chromatid segregation and the exit from mitosis1,2. Selection of APC/C targets is achieved through recognition of destruction motifs, predominantly the destruction (D)-box3 and KEN (Lys-Glu-Asn)-box4. Although this process is known to involve a co-activator protein (either Cdc20 or Cdh1) together with core APC/C subunits1,2, the structural basis for substrate recognition and ubiquitylation is not understood. Here we investigate budding yeast APC/C using single-particle electron microscopy and determine a cryo-electron microscopy map of APC/C in complex with the Cdh1 co-activator protein (APC/CCdh1) bound to a D-box peptide at 10 Å resolution. We find that a combined catalytic and substrate-recognition module is located within the central cavity of the APC/C assembled from Cdh1, Apc10—a core APC/C subunit previously implicated in substrate recognition5,6,7—and the cullin domain of Apc2. Cdh1 and Apc10, identified from difference maps, create a co-receptor for the D-box following repositioning of Cdh1 towards Apc10. Using NMR spectroscopy we demonstrate specific D-box–Apc10 interactions, consistent with a role for Apc10 in directly contributing towards D-box recognition by the APC/CCdh1 complex. Our results rationalize the contribution of both co-activator and core APC/C subunits to D-box recognition8,9 and provide a structural framework for understanding mechanisms of substrate recognition and catalysis by the APC/C.

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Figure 1: Negative-stain EM reconstructions of budding yeast APC/C show that substrate binding to APC/C Cdh1 involves Cdh1 and Apc10.
Figure 2: Cryo-EM reconstruction of budding yeast APC/C Cdh1–D-box reveals the lattice-like architecture of the complex.
Figure 3: Cdh1, Apc10, Apc2 and Apc11 form a substrate-recognition catalytic module.
Figure 4: 1 H- 15 N HSQC spectra of Apc10.

Accession codes

Primary accessions

EMBL/GenBank/DDBJ

Data deposits

EM maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-1815 (cryo-EM APC/CCdh1–D-box), EMD-1816 (apo APC/C), EMD-1817 (APC/CCdh1), EMD-1818 (APC/CCdh1–KEN-box) and EMD-1819 (APC/CCdh1–Hsl1).

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Acknowledgements

This work was funded by a Cancer Research UK grant to D.B. We thank F. Beuron for help with the early stages of this project and for EM support. We are grateful to J. Kirkpatrick for the use of the facilities of the UCL/Birkbeck Institute of Structural Molecular Biology (ISMB) Biomolecular NMR Centre.

Author information

All authors contributed to experimental design, data analysis and manuscript preparation. P.C.A.d.F. and E.H.K. collected and analysed EM data, E.H.K. prepared APC/C samples and performed ubiquitylation assays. P.C.A.d.F. determined the three-dimensional EM reconstructions and fitted coordinates. M.A.W. performed NMR experiments and analysed NMR data. E.P.M. helped collect and analyse EM data.

Correspondence to David Barford.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-12 with legends, Supplementary Tables 1-2 and additional references. (PDF 27933 kb)

Supplementary Movie 1

The movie shows morphing between APC/CCdh1 binary complex and APC/CCdh1•D-box ternary complex illustrating repositioning of Cdh1 (magenta) towards Apc10 (blue). Both complexes used are negative stain reconstructions. (AVI 6803 kb)

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da Fonseca, P., Kong, E., Zhang, Z. et al. Structures of APC/CCdh1 with substrates identify Cdh1 and Apc10 as the D-box co-receptor. Nature 470, 274–278 (2011) doi:10.1038/nature09625

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