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.

  • Article
  • Published:

Electron microscopy structure of human APC/CCDH1–EMI1 reveals multimodal mechanism of E3 ligase shutdown

Nature Structural & Molecular Biology volume 20, pages 827–835 (2013)Cite this article

Subjects

Abstract

The anaphase-promoting complex/cyclosome (APC/C) is a ~1.5-MDa multiprotein E3 ligase enzyme that regulates cell division by promoting timely ubiquitin-mediated proteolysis of key cell-cycle regulatory proteins. Inhibition of human APC/CCDH1 during interphase by early mitotic inhibitor 1 (EMI1) is essential for accurate coordination of DNA synthesis and mitosis. Here, we report a hybrid structural approach involving NMR, electron microscopy and enzymology, which reveal that EMI1's 143-residue C-terminal domain inhibits multiple APC/CCDH1 functions. The intrinsically disordered D-box, linker and tail elements, together with a structured zinc-binding domain, bind distinct regions of APC/CCDH1 to synergistically both block the substrate-binding site and inhibit ubiquitin-chain elongation. The functional importance of intrinsic structural disorder is explained by enabling a small inhibitory domain to bind multiple sites to shut down various functions of a 'molecular machine' nearly 100 times its size.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The APC/C inhibitory domain of EMI1 contains two intrinsically disordered segments separated by a zinc-dependent folded domain.
Figure 2: EM structures of APC/CCDH1 inhibited by EMI1–SKP1 and the inhibitory C-terminal domain (EMI1DLZT).
Figure 3: EMI1 is a tight binding inhibitor of APC/C Ub ligation and Ub-chain formation.
Figure 4: EMI1DLZT elements synergize to mediate optimal inhibition.
Figure 5: NMR structure of the ZBR and identification of a surface required for inhibition.
Figure 6: The EMI1 C-terminal tail is a specific inhibitor of APC/C- and UBE2S-dependent ubiquitin-chain formation.
Figure 7: Mechanisms of APC/C inhibition model for EMI1 inhibition of APC/CCDH1.

Similar content being viewed by others

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Deshaies, R.J. & Joazeiro, C.A. RING domain E3 ubiquitin ligases. Annu. Rev. Biochem. 78, 399–434 (2009).

    Article  CAS  Google Scholar 

  2. Barford, D. Structural insights into anaphase-promoting complex function and mechanism. Phil. Trans. R. Soc. Lond. B 366, 3605–3624 (2011).

    Article  CAS  Google Scholar 

  3. McLean, J.R., Chaix, D., Ohi, M.D. & Gould, K.L. State of the APC/C: organization, function, and structure. Crit. Rev. Biochem. Mol. Biol. 46, 118–136 (2011).

    Article  CAS  Google Scholar 

  4. da Fonseca, P.C. et al. Structures of APC/C(Cdh1) with substrates identify Cdh1 and Apc10 as the D-box co-receptor. Nature 470, 274–278 (2011).

    Article  CAS  Google Scholar 

  5. Chao, W.C., Kulkarni, K., Zhang, Z., Kong, E.H. & Barford, D. Structure of the mitotic checkpoint complex. Nature 484, 208–213 (2012).

    Article  CAS  Google Scholar 

  6. Buschhorn, B.A. et al. Substrate binding on the APC/C occurs between the coactivator Cdh1 and the processivity factor Doc1. Nat. Struct. Mol. Biol. 18, 6–13 (2011).

    Article  CAS  Google Scholar 

  7. Tian, W. et al. Structural analysis of human Cdc20 supports multisite degron recognition by APC/C. Proc. Natl. Acad. Sci. USA 109, 18419–18424 (2012).

    Article  CAS  Google Scholar 

  8. Musacchio, A. Spindle assembly checkpoint: the third decade. Phil. Trans. R. Soc. Lond. B 366, 3595–3604 (2011).

    Article  CAS  Google Scholar 

  9. Kim, S. & Yu, H. Mutual regulation between the spindle checkpoint and APC/C. Semin. Cell Dev. Biol. 22, 551–558 (2011).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Dong, X. et al. Control of G1 in the developing Drosophila eye: rca1 regulates Cyclin A. Genes Dev. 11, 94–105 (1997).

    Article  CAS  Google Scholar 

  12. Reimann, J.D., Gardner, B.E., Margottin-Goguet, F. & Jackson, P.K. Emi1 regulates the anaphase-promoting complex by a different mechanism than Mad2 proteins. Genes Dev. 15, 3278–3285 (2001).

    Article  CAS  Google Scholar 

  13. Hsu, J.Y., Reimann, J.D., Sorensen, C.S., Lukas, J. & Jackson, P.K. E2F-dependent accumulation of hEmi1 regulates S phase entry by inhibiting APC(Cdh1). Nat. Cell Biol. 4, 358–366 (2002).

    Article  CAS  Google Scholar 

  14. Margottin-Goguet, F. et al. Prophase destruction of Emi1 by the SCF(betaTrCP/Slimb) ubiquitin ligase activates the anaphase promoting complex to allow progression beyond prometaphase. Dev. Cell 4, 813–826 (2003).

    Article  CAS  Google Scholar 

  15. Grosskortenhaus, R. & Sprenger, F. Rca1 inhibits APC-Cdh1(Fzr) and is required to prevent cyclin degradation in G2. Dev. Cell 2, 29–40 (2002).

    Article  CAS  Google Scholar 

  16. Di Fiore, B. & Pines, J. Emi1 is needed to couple DNA replication with mitosis but does not regulate activation of the mitotic APC/C. J. Cell Biol. 177, 425–437 (2007).

    Article  CAS  Google Scholar 

  17. Machida, Y.J. & Dutta, A. The APC/C inhibitor, Emi1, is essential for prevention of rereplication. Genes Dev. 21, 184–194 (2007).

    Article  CAS  Google Scholar 

  18. Ban, K.H. et al. The END network couples spindle pole assembly to inhibition of the anaphase-promoting complex/cyclosome in early mitosis. Dev. Cell 13, 29–42 (2007).

    Article  CAS  Google Scholar 

  19. Reimann, J.D. et al. Emi1 is a mitotic regulator that interacts with Cdc20 and inhibits the anaphase promoting complex. Cell 105, 645–655 (2001).

    Article  CAS  Google Scholar 

  20. Guardavaccaro, D. et al. Control of meiotic and mitotic progression by the F box protein beta-Trcp1 in vivo. Dev. Cell 4, 799–812 (2003).

    Article  CAS  Google Scholar 

  21. Hansen, D.V., Loktev, A.V., Ban, K.H. & Jackson, P.K. Plk1 regulates activation of the anaphase promoting complex by phosphorylating and triggering SCFbetaTrCP-dependent destruction of the APC Inhibitor Emi1. Mol. Biol. Cell 15, 5623–5634 (2004).

    Article  CAS  Google Scholar 

  22. Miller, J.J. et al. Emi1 stably binds and inhibits the anaphase-promoting complex/cyclosome as a pseudosubstrate inhibitor. Genes Dev. 20, 2410–2420 (2006).

    Article  CAS  Google Scholar 

  23. Ohe, M. et al. Emi2 inhibition of the anaphase-promoting complex/cyclosome absolutely requires Emi2 binding via the C-terminal RL tail. Mol. Biol. Cell 21, 905–913 (2010).

    Article  CAS  Google Scholar 

  24. Tang, W. et al. Emi2-mediated inhibition of E2-substrate ubiquitin transfer by the anaphase-promoting complex/cyclosome through a D-box-independent mechanism. Mol. Biol. Cell 21, 2589–2597 (2010).

    Article  CAS  Google Scholar 

  25. Dunker, A.K. et al. Intrinsically disordered protein. J. Mol. Graph. Model. 19, 26–59 (2001).

    Article  CAS  Google Scholar 

  26. Gieffers, C., Dube, P., Harris, J.R., Stark, H. & Peters, J.M. Three-dimensional structure of the anaphase-promoting complex. Mol. Cell 7, 907–913 (2001).

    Article  CAS  Google Scholar 

  27. 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).

    Article  CAS  Google Scholar 

  28. Rodrigo-Brenni, M.C. & Morgan, D.O. Sequential E2s drive polyubiquitin chain assembly on APC targets. Cell 130, 127–139 (2007).

    Article  CAS  Google Scholar 

  29. Aristarkhov, A. et al. E2-C, a cyclin-selective ubiquitin carrier protein required for the destruction of mitotic cyclins. Proc. Natl. Acad. Sci. USA 93, 4294–4299 (1996).

    Article  CAS  Google Scholar 

  30. Yu, H., King, R.W., Peters, J.M. & Kirschner, M.W. Identification of a novel ubiquitin-conjugating enzyme involved in mitotic cyclin degradation. Curr. Biol. 6, 455–466 (1996).

    Article  CAS  Google Scholar 

  31. Summers, M.K., Pan, B., Mukhyala, K. & Jackson, P.K. The unique N terminus of the UbcH10 E2 enzyme controls the threshold for APC activation and enhances checkpoint regulation of the APC. Mol. Cell 31, 544–556 (2008).

    Article  CAS  Google Scholar 

  32. Dimova, N.V. et al. APC/C-mediated multiple monoubiquitylation provides an alternative degradation signal for cyclin B1. Nat. Cell Biol. 14, 168–176 (2012).

    Article  CAS  Google Scholar 

  33. Garnett, M.J. et al. UBE2S elongates ubiquitin chains on APC/C substrates to promote mitotic exit. Nat. Cell Biol. 11, 1363–1369 (2009).

    Article  CAS  Google Scholar 

  34. Williamson, A. et al. Identification of a physiological E2 module for the human anaphase-promoting complex. Proc. Natl. Acad. Sci. USA 106, 18213–18218 (2009).

    Article  CAS  Google Scholar 

  35. Wu, T. et al. UBE2S drives elongation of K11-linked ubiquitin chains by the anaphase-promoting complex. Proc. Natl. Acad. Sci. USA 107, 1355–1360 (2010).

    Article  CAS  Google Scholar 

  36. Meyer, H.J. & Rape, M. Processive ubiquitin chain formation by the anaphase-promoting complex. Semin. Cell Dev. Biol. 22, 544–550 (2011).

    Article  CAS  Google Scholar 

  37. Uzunova, K. et al. APC15 mediates CDC20 autoubiquitylation by APC/C(MCC) and disassembly of the mitotic checkpoint complex. Nat. Struct. Mol. Biol. 19, 1116–1123 (2012).

    Article  CAS  Google Scholar 

  38. Zeng, X. & King, R.W. An APC/C inhibitor stabilizes cyclin B1 by prematurely terminating ubiquitination. Nat. Chem. Biol. 8, 383–392 (2012).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  40. Morrison, J.F. Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors. Biochim. Biophys. Acta 185, 269–286 (1969).

    Article  CAS  Google Scholar 

  41. 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).

    Article  CAS  Google Scholar 

  42. Pashkova, N. et al. WD40 repeat propellers define a ubiquitin-binding domain that regulates turnover of F box proteins. Mol. Cell 40, 433–443 (2010).

    Article  CAS  Google Scholar 

  43. Wickliffe, K.E., Lorenz, S., Wemmer, D.E., Kuriyan, J. & Rape, M. The mechanism of linkage-specific ubiquitin chain elongation by a single-subunit E2. Cell 144, 769–781 (2011).

    Article  CAS  Google Scholar 

  44. Baboshina, O.V. & Haas, A.L. Novel multiubiquitin chain linkages catalyzed by the conjugating enzymes E2EPF and RAD6 are recognized by 26 S proteasome subunit 5. J. Biol. Chem. 271, 2823–2831 (1996).

    Article  CAS  Google Scholar 

  45. Sironi, L. et al. Crystal structure of the tetrameric Mad1-Mad2 core complex: implications of a 'safety belt' binding mechanism for the spindle checkpoint. EMBO J. 21, 2496–2506 (2002).

    Article  CAS  Google Scholar 

  46. Luo, X., Tang, Z., Rizo, J. & Yu, H. The Mad2 spindle checkpoint protein undergoes similar major conformational changes upon binding to either Mad1 or Cdc20. Mol. Cell 9, 59–71 (2002).

    Article  Google Scholar 

  47. Dyson, H.J. & Wright, P.E. Intrinsically unstructured proteins and their functions. Nat. Rev. Mol. Cell Biol. 6, 197–208 (2005).

    Article  CAS  Google Scholar 

  48. Reimann, J.D. & Jackson, P.K. Emi1 is required for cytostatic factor arrest in vertebrate eggs. Nature 416, 850–854 (2002).

    Article  CAS  Google Scholar 

  49. Moshe, Y., Bar-On, O., Ganoth, D. & Hershko, A. Regulation of the action of early mitotic inhibitor 1 on the anaphase-promoting complex/cyclosome by cyclin-dependent kinases. J. Biol. Chem. 286, 16647–16657 (2011).

    Article  CAS  Google Scholar 

  50. Kjaergaard, M. & Poulsen, F.M. Sequence correction of random coil chemical shifts: correlation between neighbor correction factors and changes in the Ramachandran distribution. J. Biomol. NMR 50, 157–165 (2011).

    Article  CAS  Google Scholar 

  51. Bieniossek, C., Richmond, T.J. & Berger, I. MultiBac: multigene baculovirus-based eukaryotic protein complex production. Curr. Protoc. Protein Sci. 51, 5.20 (2008).

    Google Scholar 

  52. Keller, R.L.J. The computer aided resonance assignment tutorial. 〈http://cara.nmr-software.org/downloads/3-85600-112-3.pdf〉 (CANTINA Verlag, Zurich, 2004).

  53. Güntert, P., Mumenthaler, C. & Wuthrich, K. Torsion angle dynamics for NMR structure calculation with the new program DYANA. J. Mol. Biol. 273, 283–298 (1997).

    Article  Google Scholar 

  54. Shen, Y., Delaglio, F., Cornilescu, G. & Bax, A. TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J. Biomol. NMR 44, 213–223 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to C.-G. Park, D. King, R. Pappu, B. Dye, C. Rock, P. Rodrigues and R. Cassell for advice and/or assistance. N.G.B. is a fellow of the Jane Coffin Childs Memorial Fund for Medical Research. The laboratory of R.W.K. was supported by American Lebanese Syrian Associated Charities (ALSAC), US National Institutes of Health (NIH) P30CA021765, R01CA082491 and 1R01GM08315. The laboratory of H.S. was supported by Deutsche Forschungsgemeinschaft Sonderforschungsbereich 860. The laboratory of J.-M.P. was supported by Boehringer Ingelheim, the Austrian Science Fund (FWF special research program SFB F34 'Chromosome Dynamics', grant W1221 'DK: Structure and Interaction of Biological Macromolecules' and Wittgenstein award Z196-B20), the Austrian Research Promotion Agency (FFG, Laura Bassi Center for Optimized Structural Studies), the Vienna Science and Technology Fund (WWTF LS09-13) and the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 241548 (MitoSys). The laboratory of B.A.S. was supported by ALSAC, NIH P30CA021765, R01GM065930 and the Howard Hughes Medical Institute. B.A.S. is an HHMI investigator.

Author information

Author notes

  1. Jeremiah J Frye, Nicholas G Brown and Georg Petzold: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, Tennessee

    Jeremiah J Frye, Nicholas G Brown, Edmond R Watson, Christy R R Grace, Richard W Kriwacki & Brenda A Schulman

  2. Research Institute of Molecular Pathology, Vienna, Austria

    Georg Petzold, Marc A Jarvis & Jan-Michael Peters

  3. Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Sciences Center, Memphis, Tennessee

    Edmond R Watson, Richard W Kriwacki & Brenda A Schulman

  4. Hartwell Center for Bioinformatics and Biotechnology, St. Jude Children's Research Hospital, Memphis, Tennessee

    Amanda Nourse

  5. Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Holger Stark

  6. Department of 3D Electron Cryomicroscopy, Institute of Microbiology and Genetics, Georg-August Universität, Göttingen, Germany

    Holger Stark

  7. Howard Hughes Medical Institute, St. Jude Children's Research Hospital, Memphis, Tennessee

    Brenda A Schulman

Authors
  1. Jeremiah J Frye
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nicholas G Brown
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Georg Petzold
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Edmond R Watson
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Christy R R Grace
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Amanda Nourse
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Marc A Jarvis
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Richard W Kriwacki
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jan-Michael Peters
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Holger Stark
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Brenda A Schulman
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.-M.P., H.S. and B.A.S. planned and supervised the project. J.J.F., N.G.B., G.P., E.R.W., C.R.R.G., A.N. and M.A.J. designed the experiments. G.P. prepared samples for and contributed to EM experiments. J.J.F., N.G.B. and E.R.W. performed biochemical and biophysical analyses. A.N. performed analytical ultracentrifugation. C.R.R.G. and R.W.K. performed NMR analyses. H.S. performed EM. J.J.F., N.G.B., E.R.W., H.S. and B.A.S. prepared the manuscript with input from all authors.

Corresponding authors

Correspondence to Jan-Michael Peters, Holger Stark or Brenda A Schulman.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 7216 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Frye, J., Brown, N., Petzold, G. et al. Electron microscopy structure of human APC/CCDH1–EMI1 reveals multimodal mechanism of E3 ligase shutdown. Nat Struct Mol Biol 20, 827–835 (2013). https://doi.org/10.1038/nsmb.2593

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2593

This article is cited by

Search

Advanced 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