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.

Molecular architecture and mechanism of the anaphase-promoting complex

Nature volume 513, pages 388–393 (2014)Cite this article

Subjects

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.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

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

Figure 1: Electron microscopy reconstructions of the H. sapiens APC/C–CDH1–HSL1 ternary complex at 7.4 Å resolution.
Figure 2: The TPR homodimers assemble to form a quasi-symmetric array.
Figure 3: Architecture of platform subunits.
Figure 4: Mechanism of D box recognition by CDH1 and APC10 and interactions with APC/C subunits.
Figure 5: Conformational change of the APC/C induced by CDH1NTD.

Similar content being viewed by others

Accession codes

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).

References

  1. Teixeira, L. K. & Reed, S. I. Ubiquitin ligases and cell cycle control. Annu. Rev. Biochem. 82, 387–414 (2013)

    Article  CAS  PubMed  Google Scholar 

  2. Pines, J. Cubism and the cell cycle: the many faces of the APC/C. Nature Rev. Mol. Cell Biol. 12, 427–438 (2011)

    Article  CAS  Google Scholar 

  3. Primorac, I. & Musacchio, A. Panta rhei: the APC/C at steady state. J. Cell Biol. 201, 177–189 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Lara-Gonzalez, P., Westhorpe, F. G. & Taylor, S. S. The spindle assembly checkpoint. Curr. Biol. 22, R966–R980 (2012)

    Article  CAS  PubMed  Google Scholar 

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

    Article  ADS  CAS  PubMed  Google Scholar 

  6. 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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Kimata, Y., Baxter, J. E., Fry, A. M. & Yamano, H. A role for the Fizzy/Cdc20 family of proteins in activation of the APC/C distinct from substrate recruitment. Mol. Cell 32, 576–583 (2008)

    Article  CAS  PubMed  Google Scholar 

  8. Schreiber, A. et al. Structural basis for the subunit assembly of the anaphase-promoting complex. Nature 470, 227–232 (2011)

    Article  ADS  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Zhang, Z. et al. Recombinant expression, reconstitution and structure of human anaphase-promoting complex (APC/C). Biochem. J. 449, 365–371 (2013)

    Article  CAS  PubMed  Google Scholar 

  11. 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  PubMed  PubMed Central  Google Scholar 

  12. 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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 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  PubMed  Google Scholar 

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

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  15. 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  ADS  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Wang, J., Dye, B. T., Rajashankar, K. R., Kurinov, I. & Schulman, B. A. Insights into anaphase promoting complex TPR subdomain assembly from a CDC26–APC6 structure. Nature Struct. Mol. Biol. 16, 987–989 (2009)

    Article  CAS  Google Scholar 

  19. Zhang, Z., Kulkarni, K., Hanrahan, S. J., Thompson, A. J. & Barford, D. The APC/C subunit Cdc16/Cut9 is a contiguous tetratricopeptide repeat superhelix with a homo-dimer interface similar to Cdc27. EMBO J. 29, 3733–3744 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Zhang, Z. et al. The four canonical TPR subunits of human APC/C form related homo-dimeric structures and stack in parallel to form a TPR suprahelix. J. Mol. Biol. 425, 4236–4248 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Hutchins, J. R. et al. Systematic analysis of human protein complexes identifies chromosome segregation proteins. Science 328, 593–599 (2010)

    Article  ADS  MathSciNet  CAS  PubMed  PubMed Central  Google Scholar 

  22. He, J. et al. The structure of the 26S proteasome subunit Rpn2 reveals its PC repeat domain as a closed toroid of two concentric alpha-helical rings. Structure 20, 513–521 (2012)

    Article  CAS  PubMed  Google Scholar 

  23. Hall, M. C., Torres, M. P., Schroeder, G. K. & Borchers, C. H. Mnd2 and Swm1 are core subunits of the Saccharomyces cerevisiae anaphase-promoting complex. J. Biol. Chem. 278, 16698–16705 (2003)

    Article  CAS  PubMed  Google Scholar 

  24. Duda, D. M. et al. Structural insights into NEDD8 activation of cullin-RING ligases: conformational control of conjugation. Cell 134, 995–1006 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Calabrese, M. F. et al. A RING E3-substrate complex poised for ubiquitin-like protein transfer: structural insights into cullin-RING ligases. Nature Struct. Mol. Biol. 18, 947–949 (2011)

    Article  CAS  Google Scholar 

  26. He, J. et al. Insights into degron recognition by APC/C coactivators from the structure of an Acm1-Cdh1 complex. Mol. Cell 50, 649–660 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 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)

    Article  CAS  PubMed  Google Scholar 

  28. 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)

    Article  CAS  PubMed  Google Scholar 

  29. 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)

    Article  CAS  PubMed  Google Scholar 

  30. 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)

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 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)

    Article  CAS  PubMed  Google Scholar 

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

    Article  ADS  CAS  PubMed  Google Scholar 

  34. 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  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  35. Izawa, D. & Pines, J. How APC/C-Cdc20 changes its substrate specificity in mitosis. Nature Cell Biol. 13, 223–233 (2011)

    Article  CAS  PubMed  Google Scholar 

  36. Dou, H., Buetow, L., Sibbet, G. J., Cameron, K. & Huang, D. T. BIRC7–E2 ubiquitin conjugate structure reveals the mechanism of ubiquitin transfer by a RING dimer. Nature Struct. Mol. Biol. 19, 876–883 (2012)

    Article  CAS  Google Scholar 

  37. Plechanovová, A., Jaffray, E. G., Tatham, M. H., Naismith, J. H. & Hay, R. T. Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis. Nature 489, 115–120 (2012)

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  38. Pruneda, J. N. et al. Structure of an E3:E2∼Ub complex reveals an allosteric mechanism shared among RING/U-box ligases. Mol. Cell 47, 933–942 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Saha, A., Lewis, S., Kleiger, G., Kuhlman, B. & Deshaies, R. J. Essential role for ubiquitin-ubiquitin-conjugating enzyme interaction in ubiquitin discharge from Cdc34 to substrate. Mol. Cell 42, 75–83 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. 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  PubMed  PubMed Central  Google Scholar 

  41. Das, R. et al. Allosteric activation of E2-RING finger-mediated ubiquitylation by a structurally defined specific E2-binding region of gp78. Mol. Cell 34, 674–685 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. 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  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  43. 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  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  44. Wang, W. & Kirschner, M. W. Emi1 preferentially inhibits ubiquitin chain elongation by the anaphase-promoting complex. Nature Cell Biol. 15, 797–806 (2013)

    Article  CAS  PubMed  Google Scholar 

  45. Saha, A. & Deshaies, R. J. Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation. Mol. Cell 32, 21–31 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Jin, L., Williamson, A., Banerjee, S., Philipp, I. & Rape, M. Mechanism of ubiquitin-chain formation by the human anaphase-promoting complex. Cell 133, 653–665 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Frye, J. J. et al. Electron microscopy structure of human APC/C(CDH1)-EMI1 reveals multimodal mechanism of E3 ligase shutdown. Nature Struct. Mol. Biol. 20, 827–835 (2013)

    Article  CAS  Google Scholar 

  48. Berger, I., Fitzgerald, D. J. & Richmond, T. J. Baculovirus expression system for heterologous multiprotein complexes. Nature Biotechnol. 22, 1583–1587 (2004)

    Article  CAS  Google Scholar 

  49. Fitzgerald, D. J. et al. Multiprotein expression strategy for structural biology of eukaryotic complexes. Structure 15, 275–279 (2007)

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  51. Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007)

    Article  CAS  PubMed  Google Scholar 

  52. Mindell, J. A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003)

    Article  PubMed  Google Scholar 

  53. Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003)

    Article  CAS  PubMed  Google Scholar 

  55. Scheres, S. H. & Chen, S. Prevention of overfitting in cryo-EM structure determination. Nature Methods 9, 853–854 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Han, D. et al. Crystal structure of the N-terminal domain of anaphase-promoting complex subunit 7. J. Biol. Chem. 284, 15137–15146 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. 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)

    Article  CAS  PubMed  Google Scholar 

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

    Article  ADS  CAS  PubMed  Google Scholar 

  59. Roy, A., Kucukural, A. & Zhang, Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nature Protocols 5, 725–738 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Sutton, R. B., Fasshauer, D., Jahn, R. & Brunger, A. T. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution. Nature 395, 347–353 (1998)

    Article  ADS  CAS  PubMed  Google Scholar 

  61. 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)

    Article  CAS  PubMed  Google Scholar 

  62. Yang, Z. et al. UCSF Chimera, MODELLER, and IMP: an integrated modeling system. J. Struct. Biol. 179, 269–278 (2012)

    Article  CAS  PubMed  Google Scholar 

  63. Trabuco, L. G., Villa, E., Schreiner, E., Harrison, C. B. & Schulten, K. Molecular dynamics flexible fitting: a practical guide to combine cryo-electron microscopy and X-ray crystallography. Methods 49, 174–180 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38. 27–38 (1996)

    Article  CAS  PubMed  Google Scholar 

  65. Phillips, J. C. et al. Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781–1802 (2005)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  CAS  PubMed  Google Scholar 

  67. Li, Y. & Sousa, R. Expression and purification of E. coli BirA biotin ligase for in vitro biotinylation. Protein Expr. Purif. 82, 162–167 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. 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  PubMed  PubMed Central  Google Scholar 

Download references

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

  1. Leifu Chang, Ziguo Zhang, Jing Yang & David Barford

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

  2. Leifu Chang and Ziguo Zhang: These authors contributed equally to this work.

Authors and 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

Authors
  1. Leifu Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ziguo Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jing Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stephen H. McLaughlin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. David Barford
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding author

Correspondence to David Barford.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 APC/C complexes used in this study.

a, Coomassie blue stained SDS gel of the human APC/C–CDH1–HSL1 ternary complex. b, Silver stained SDS gel of apo APC/C and APC/CΔAPC15. c, Coomassie blue stained SDS gel of apo APC/C and APC/CΔRING. d, Cryo-electron microscopy micrograph of APC/C–CDH1–HSL1 ternary complex. e, Cryo-electron microscopy micrograph of apo APC/C (WT). f, Cryo-electron microscopy micrograph of APC/CΔRING. g, Cryo-electron microscopy micrograph of APC/CΔAPC15.

Extended Data Figure 2 Electron microscopy reconstructions of APC/C complexes described in this study.

a, Human APC/C–CDH1–HSL1 ternary complex. b, Apo APC/C. c, APC/CΔRING. d, APC/CΔAPC15. e, Fourier shell correlation (FSC) plots for four complexes. The resolution is based on the ‘Gold Standard’ criterion of an FSC at 0.143 (refs 54, 55). Also shown is the FSC plot between the atomic model and cryo electron microscopy map of APC/C–CDH1–HSL1.

Extended Data Figure 3 Structures of APC1, APC4, APC6, CDH1 and APC10.

a, Figure to show fitting of S. pombe APC6–APC12 (Cut9–Hcn26) crystal structure19 into the segmented APC6 electron microscopy density of APC/C–CDH1–HSL1. b, Superimposition of APC6–APC12 before (cyan) and after fitting using MDFF (pink). c, APC13 and APC16 interact with structurally equivalent sites (i) to (vii) on seven TPR subunits created by the TPR α-helices 8B, 9A and 9B. Shown are six sites (i) to (iii) (APC16) and (v) to (vii) (APC13). Roman numerals refer to interfaces labelled in Fig. 3e. d, Details of APC4WD40; e, APC1PC; f, APC1WD40. g, Electron microscopy density for CDH1; h, CDH1WD40 domain; i, APC10. j, APC10 interactions with APC1PC include the conserved 70s loop shown previously to mediate APC10 association with the APC/C32.

Extended Data Figure 4 APC4–mFab two dimensional class averages.

a, Selection of two dimensional class averages derived from negatively stained electron microscopy micrographs of APC4–mFab complexes. Two representative views of mFab are indicated by red arrows. b, Projections of the APC4 segmented density from the APC/C–CDH1–HSL1 cryo electron microscopy map match experimentally-derived two dimensional class averages of APC4.

Extended Data Figure 5 Three dimensional classes of APC/C–CDH1–HSL1.

a, The major classes were identified showing that the population consisted of 60% ternary complex and 40% apo complex (the CDH1 position is indicated with a red circle). b, Densities of APC2CTD–APC11 are visible only at lower contour level (outlined by a grey circle). Further three dimensional classification of the ternary complex into 10 classes revealed structural variability of the APC2CTD–APC11 module. In one class (Class 9), density for the entire APC2 is very weak, suggesting loss of this subunit in a small proportion (∼8%) of the total. c, Stereo-view of a superimposition of the 10 three dimensional classes. d, Although variable, APC2CTD–APC11 in the ternary complex shifts upward relative to the apo structure (yellow).

Extended Data Figure 6 APC/CCDH1 ubiquitination of HSL1 is reduced by APC10 and D box (P8 to P10) mutations.

Mutations of D box residues P8 to P10 to Ala (HSL1MutP8-10) and mutations of APC10 (S88A/N147A) (APC/CMutAPC10) at the D box-binding site reduce APC/C ubiquitination activity. Combining D box and APC10 mutations potentiated the reduction of APC/C ubiquitination activity. A contaminant band from CDH1 is indicated with an asterisk.

Extended Data Figure 7 Three dimensional classes of apo APC/C.

a, The 10 three dimensional classes of apo APC/C. b, Stereoview of a superimposition of the 10 three dimensional classes shows relatively little structural variability of the APC2CTD–APC11 module (circled).

Extended Data Figure 8 Comparison of APC/C–CDH1–HSL1 model with published APC/C electron microscopy reconstructions.

a, Human APC/C–CDH1–EMI1 (ref. 47). Density assigned to the inhibitory zinc-binding region and polybasic tail of EMI1 (EMI1ZT) is indicated. b, Human APC/CMCC (ref. 14). Density corresponding to the mitotic checkpoint complex (MCC) is indicated in a black border. This analysis shows that the activated conformation of APC/C observed in the APC/C–CDH1–HSL1 ternary complex is shared in the inhibited states of APC/C with EMI1 and the MCC (both with coactivator). The APC2CTD–APC11 module interacts with MCC and the inhibitory zinc-binding region (ZBR) and C-terminal polybasic tail of EMI1 (EMI1ZT).

Extended Data Figure 9 Surface plasmon resonance data.

a, SPR sensorgrams for ternary APC/C, apo APC/C and APC2CTD–APC11 binding to UBCH10, UBCH10–Ub, UBE2S and UBE2S-ΔC. b, Equilibrium fitting for ternary and apo APC/C association with UBCH10. The equilibrium dissociation constant (KeqD) is derived from this fit. c, Fit of the observed association rate constant (kobs) (kobs = kon[analyte] + koff) for ternary and apo APC/C association with UBCH10. d, Equilibrium fitting for ternary and apo APC/C association with UBCH10–Ub. e, Fit of the observed association rate constant (kobs) for ternary APC/C association with UBCH10–Ub. f, Equilibrium fitting for ternary and apo APC/C association with UBE2S. The reason for the lower maximum response unit (RU) at equilibrium for apo APC/C binding to UBE2S, relative to ternary APC/C, is unclear. g, Fit of the observed association rate constant (kobs) (for ternary and apo APC/C association with UBE2S. The rate for 865 nM apo APC/C was not fitted as this was close to the limit of the Biacore T200 response. h, Table summarizing APC/C–CDH1–HSL1 (ternary APC/C), apo APC/C and APC2CTD–APC11 dissociation constants for UBCH10, UBCH10–ubiquitin and UBE2S. KeqD, equilibrium dissociation constant; Kkind, kinetic dissociation constant = koff/kon. ND1, not determined because equilibrium binding was not achieved. ND2, not determined due to fast kon and koff. Standard errors of the fit are listed. ΔN-UBCH10 (deletion of residues 4–32 of UBCH10) and UBCH10 had similar relative affinities for ternary and apo APC/C (data not shown).

Extended Data Table 1 a, Description of human APC/C subunits; b, summary of electron microscopy data statistics
Full size table

Supplementary information

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. (MP4 27490 kb)

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. (MOV 7381 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chang, L., Zhang, Z., Yang, J. et al. Molecular architecture and mechanism of the anaphase-promoting complex. Nature 513, 388–393 (2014). https://doi.org/10.1038/nature13543

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature13543

This article is cited by

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

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