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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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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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.
The authors declare no competing financial interests.
The file contains Supplementary Figures 1-12 with legends, Supplementary Tables 1-2 and additional references. (PDF 27933 kb)
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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