Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

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.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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).

References

  1. 1

    Peters, J. M. The anaphase promoting complex/cyclosome: a machine designed to destroy. Nature Rev. Mol. Cell Biol. 7, 644–656 (2006)

    CAS  Article  Google Scholar 

  2. 2

    Thornton, B. R. & Toczyski, D. P. Precise destruction: an emerging picture of the APC. Genes Dev. 20, 3069–3078 (2006)

    CAS  Article  Google Scholar 

  3. 3

    Glotzer, M., Murray, A. W. & Kirschner, M. W. Cyclin is degraded by the ubiquitin pathway. Nature 349, 132–138 (1991)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Pfleger, C. M. & Kirschner, M. W. The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1. Genes Dev. 14, 655–665 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Carroll, C. W. & Morgan, D. O. The Doc1 subunit is a processivity factor for the anaphase-promoting complex. Nature Cell Biol. 4, 880–887 (2002)

    CAS  Article  Google Scholar 

  6. 6

    Passmore, L. A. et al. Doc1 mediates the activity of the anaphase-promoting complex by contributing to substrate recognition. EMBO J. 22, 786–796 (2003)

    CAS  Article  Google Scholar 

  7. 7

    Carroll, C. W., Enquist-Newman, M. & Morgan, D. O. The APC subunit Doc1 promotes recognition of the substrate destruction box. Curr. Biol. 15, 11–18 (2005)

    CAS  Article  Google Scholar 

  8. 8

    Yamano, H., Gannon, J., Mahbubani, H. & Hunt, T. Cell cycle-regulated recognition of the destruction box of cyclin B by the APC/C in Xenopus egg extracts. Mol. Cell 13, 137–147 (2004)

    CAS  Article  Google Scholar 

  9. 9

    Eytan, E., Moshe, Y., Braunstein, I. & Hershko, A. Roles of the anaphase-promoting complex/cyclosome and of its activator Cdc20 in functional substrate binding. Proc. Natl Acad. Sci. USA 103, 2081–2086 (2006)

    ADS  CAS  Article  Google Scholar 

  10. 10

    Vodermaier, H. C., Gieffers, C., Maurer-Stroh, S., Eisenhaber, F. & Peters, J. M. TPR subunits of the anaphase-promoting complex mediate binding to the activator protein CDH1. Curr. Biol. 13, 1459–1468 (2003)

    CAS  Article  Google Scholar 

  11. 11

    Kraft, C., Vodermaier, H. C., Maurer-Stroh, S., Eisenhaber, F. & Peters, J. M. The WD40 propeller domain of Cdh1 functions as a destruction box receptor for APC/C substrates. Mol. Cell 18, 543–553 (2005)

    CAS  Article  Google Scholar 

  12. 12

    Thornton, B. R. et al. An architectural map of the anaphase-promoting complex. Genes Dev. 20, 449–460 (2006)

    CAS  Article  Google Scholar 

  13. 13

    Wendt, K. S. et al. Crystal structure of the APC10/DOC1 subunit of the human anaphase-promoting complex. Nature Struct. Biol. 8, 784–788 (2001)

    CAS  Article  Google Scholar 

  14. 14

    Burton, J. L. & Solomon, M. J. D box and KEN box motifs in budding yeast Hsl1p are required for APC-mediated degradation and direct binding to Cdc20p and Cdh1p. Genes Dev. 15, 2381–2395 (2001)

    CAS  Article  Google Scholar 

  15. 15

    Hilioti, Z., Chung, Y. S., Mochizuki, Y., Hardy, C. F. & Cohen-Fix, O. The anaphase inhibitor Pds1 binds to the APC/C-associated protein Cdc20 in a destruction box-dependent manner. Curr. Biol. 11, 1347–1352 (2001)

    CAS  Article  Google Scholar 

  16. 16

    Pfleger, C. M., Lee, E. & Kirschner, M. W. Substrate recognition by the Cdc20 and Cdh1 components of the anaphase-promoting complex. Genes Dev. 15, 2396–2407 (2001)

    CAS  Article  Google Scholar 

  17. 17

    Schwab, M., Neutzner, M., Mocker, D. & Seufert, W. Yeast Hct1 recognizes the mitotic cyclin Clb2 and other substrates of the ubiquitin ligase APC. EMBO J. 20, 5165–5175 (2001)

    CAS  Article  Google Scholar 

  18. 18

    Passmore, L. A. et al. Structural analysis of the anaphase-promoting complex reveals multiple active sites and insights into polyubiquitylation. Mol. Cell 20, 855–866 (2005)

    CAS  Article  Google Scholar 

  19. 19

    Dube, P. et al. Localization of the coactivator Cdh1 and the cullin subunit Apc2 in a cryo-electron microscopy model of vertebrate APC/C. Mol. Cell 20, 867–879 (2005)

    CAS  Article  Google Scholar 

  20. 20

    Ohi, M. D. et al. Structural organization of the anaphase-promoting complex bound to the mitotic activator Slp1. Mol. Cell 28, 871–885 (2007)

    CAS  Article  Google Scholar 

  21. 21

    Herzog, F. et al. Structure of the anaphase-promoting complex/cyclosome interacting with a mitotic checkpoint complex. Science 323, 1477–1481 (2009)

    ADS  CAS  Article  Google Scholar 

  22. 22

    Au, S. W., Leng, X., Harper, J. W. & Barford, D. Implications for the ubiquitination reaction of the anaphase-promoting complex from the crystal structure of the Doc1/Apc10 subunit. J. Mol. Biol. 316, 955–968 (2002)

    CAS  Article  Google Scholar 

  23. 23

    Matyskiela, M. E. & Morgan, D. O. Analysis of activator-binding sites on the APC/C supports a cooperative substrate-binding mechanism. Mol. Cell 34, 68–80 (2009)

    CAS  Article  Google Scholar 

  24. 24

    Yamano, H., Tsurumi, C., Gannon, J. & Hunt, T. The role of the destruction box and its neighbouring lysine residues in cyclin B for anaphase ubiquitin-dependent proteolysis in fission yeast: defining the D-box receptor. EMBO J. 17, 5670–5678 (1998)

    CAS  Article  Google Scholar 

  25. 25

    Zheng, N. et al. Structure of the Cul1–Rbx1–Skp1–F boxSkp2 SCF ubiquitin ligase complex. Nature 416, 703–709 (2002)

    ADS  CAS  Article  Google Scholar 

  26. 26

    Zhang, Z. et al. Molecular structure of the N-terminal domain of the APC/C subunit Cdc27 reveals a homo-dimeric tetratricopeptide repeat architecture. J. Mol. Biol. 397, 1316–1328 (2010)

    CAS  Article  Google Scholar 

  27. 27

    van Heel, M. et al. Single-particle electron cryo-microscopy: towards atomic resolution. Q. Rev. Biophys. 33, 307–369 (2000)

    CAS  Article  Google Scholar 

  28. 28

    Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199 (1996)

    CAS  Article  Google Scholar 

  29. 29

    Ludtke, S. J., Baldwin, P. R. & Chiu, W. EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999)

    CAS  Article  Google Scholar 

  30. 30

    Passmore, L. A., Barford, D. & Harper, J. W. Purification and assay of the budding yeast anaphase-promoting complex. Methods Enzymol. 398, 195–219 (2005)

    CAS  Article  Google Scholar 

  31. 31

    Kelley, L. A. & Sternberg, M. J. Protein structure prediction on the web: a case study using the Phyre server. Nature Protocols 4, 363–371 (2009)

    CAS  Article  Google Scholar 

  32. 32

    Schuetz, A. et al. Structural basis for molecular recognition and presentation of histone H3 by WDR5. EMBO J. 25, 4245–4252 (2006)

    CAS  Article  Google Scholar 

  33. 33

    Angers, S. et al. Molecular architecture and assembly of the DDB1–CUL4A ubiquitin ligase machinery. Nature 443, 590–593 (2006)

    ADS  CAS  Article  Google Scholar 

  34. 34

    Navaza, J., Lepault, J., Rey, F. A., Alvarez-Rua, C. & Borge, J. On the fitting of model electron densities into EM reconstructions: a reciprocal-space formulation. Acta Crystallogr. D Biol. Crystallogr. 58, 1820–1825 (2002)

    CAS  Article  Google Scholar 

  35. 35

    Baryshnikova, O. K., Williams, T. C. & Sykes, B. D. Internal pH indicators for biomolecular NMR. J. Biomol. NMR 41, 5–7 (2008)

    CAS  Article  Google Scholar 

  36. 36

    Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995)

    CAS  Article  Google Scholar 

  37. 37

    Vranken, W. F. et al. The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins 59, 687–696 (2005)

    CAS  Article  Google Scholar 

  38. 38

    Wu, D., Chen, A. & Johnson, C. S., Jr An improved diffusion-ordered spectroscopy experiment incorporating bipolar-gradient pulses. J. Magn. Reson. A 115, 260–264 (1995)

    ADS  CAS  Article  Google Scholar 

Download references

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

Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to David Barford.

Ethics declarations

Competing interests

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)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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). https://doi.org/10.1038/nature09625

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing