Letter | Published:

Structure of the apoptotic protease-activating factor 1 bound to ADP

Nature volume 434, pages 926933 (14 April 2005) | Download Citation

Subjects

Abstract

Apoptosis is executed by caspases, which undergo proteolytic activation in response to cell death stimuli1. The apoptotic protease-activating factor 1 (Apaf-1) controls caspase activation downstream of mitochondria2. During apoptosis, Apaf-1 binds to cytochrome c and in the presence of ATP/dATP forms an apoptosome, leading to the recruitment and activation of the initiator caspase, caspase-9 (ref. 2). The mechanisms underlying Apaf-1 function are largely unknown. Here we report the 2.2-Å crystal structure of an ADP-bound, WD40-deleted Apaf-1, which reveals the molecular mechanism by which Apaf-1 exists in an inactive state before ATP binding. The amino-terminal caspase recruitment domain packs against a three-layered α/β fold, a short helical motif and a winged-helix domain, resulting in the burial of the caspase-9-binding interface. The deeply buried ADP molecule serves as an organizing centre to strengthen interactions between these four adjoining domains, thus locking Apaf-1 in an inactive conformation. Apaf-1 binds to and hydrolyses ATP/dATP and their analogues. The binding and hydrolysis of nucleotides seem to drive conformational changes that are essential for the formation of the apoptosome and the activation of caspase-9.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    & Molecular mechanisms of caspase regulation during apoptosis. Nature Rev. Mol. Cell Biol. 5, 897–907 (2004)

  2. 2.

    The expanding role of mitochondria in apoptosis. Genes Dev. 15, 2922–2933 (2001)

  3. 3.

    et al. Cytochrome c and dATP-dependent formation of Apaf-1/Caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479–489 (1997)

  4. 4.

    , , & An APAF-1-cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J. Biol. Chem. 274, 11549–11556 (1999)

  5. 5.

    & Cytochrome c promotes caspase-9 activation by inducing nucleotide binding to Apaf-1. J. Biol. Chem. 275, 31199–31203 (2000)

  6. 6.

    , , & Role of cytochrome c and dATP/ATP hydrolysis in Apaf-1-mediated caspase-9 activation and apoptosis. EMBO J. 18, 3586–3595 (1999)

  7. 7.

    , , , & Cytochrome c and dATP-mediated oligomerization of Apaf-1 is a prerequisite for procaspase-9 activation. J. Biol. Chem. 274, 17941–17945 (1999)

  8. 8.

    & Caspase-9 and Apaf-1 form an active holoenzyme. Genes Dev. 13, 3179–3184 (1999)

  9. 9.

    & The NOD: a signaling module that regulates apoptosis and host defense against pathogens. Oncogene 20, 6473–6481 (2001)

  10. 10.

    , , & WD-40 repeat region regulates Apaf-1 self-association and procaspase-9 activation. J. Biol. Chem. 273, 33489–33494 (1998)

  11. 11.

    , , & Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol. Cell 1, 949–957 (1998)

  12. 12.

    & Five years on the wings of fork head. Mech. Dev. 57, 3–20 (1996)

  13. 13.

    & Protein structure comparison by alignment of distance matrices. J. Mol. Biol. 233, 123–138 (1993)

  14. 14.

    , , & Crystal structure of the hexamerization domain of N-ethylmaleimide-sensitive fusion protein. Cell 94, 525–536 (1998)

  15. 15.

    et al. Structure of the AAA ATPase p97. Mol. Cell 6, 1473–1484 (2000)

  16. 16.

    & AAA proteins. Curr. Opin. Struct. Biol. 12, 746–753 (2002)

  17. 17.

    , , & ATP-activated oligomerization as a mechanism for apoptosis regulation: fold and mechanism prediction for CED-4. Proteins 39, 197–203 (2000)

  18. 18.

    et al. Structural basis of procaspase-9 recruitment by the apoptotic protease-activating factor 1. Nature 399, 547–555 (1999)

  19. 19.

    et al. Nucleotide requirements for the in vitro activation of the apoptosis protein-activating factor-1-mediated caspase pathway. J. Biol. Chem. 275, 29–34 (2000)

  20. 20.

    et al. Induction of an apoptotic program in cell-free extracts by 2-chloro-2′-deoxyadenosine 5′-triphosphate and cytochrome c. Proc. Natl Acad. Sci. USA 95, 9567–9571 (1998)

  21. 21.

    , , & Caspase activation involves the formation of the aposome, a large ( 700 kDa) caspase-activating complex. J. Biol. Chem. 274, 22686–22692 (1999)

  22. 22.

    et al. The structures of HsIU and the ATP-dependent protease HsIU-HsIV. Nature 403, 800–805 (2000)

  23. 23.

    , , , & Mechanisms of conformational change for a replicative hexameric helicase of SV40 large tumor antigen. Cell 119, 47–60 (2004)

  24. 24.

    et al. Three-dimensional structure of the apoptosome: Implications for assembly, procaspase-9 binding and activation. Mol. Cell 9, 423–432 (2002)

  25. 25.

    & Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

  26. 26.

    & Automated structure solution for MIR and MAD. Acta Crystallogr. D 55, 849–861 (1999)

  27. 27.

    , , & Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

  28. 28.

    . The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

  29. 29.

    Molscript: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991)

  30. 30.

    , & Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins Struct. Funct. Genet. 11, 281–296 (1991)

Download references

Acknowledgements

We thank M. Becker, A. Saxena, K. Dedrick and L. Walsh for assistance, A. Wist for help with the TLC, L. Gu for help with the omit map, F. Hughson for critically reading the manuscript, and members of the Shi laboratory for discussion. This work was supported by the NIH. S.J.R. is a Fellow of the Leukemia and Lymphoma Society.

Author information

Affiliations

  1. Department of Molecular Biology, Princeton University, Lewis Thomas Laboratory, Washington Road, Princeton, New Jersey 08544, USA

    • Stefan J. Riedl
    • , Wenyu Li
    • , Yang Chao
    •  & Yigong Shi
  2. Joint Center for Structural Genomics, University of California-San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA

    • Robert Schwarzenbacher

Authors

  1. Search for Stefan J. Riedl in:

  2. Search for Wenyu Li in:

  3. Search for Yang Chao in:

  4. Search for Robert Schwarzenbacher in:

  5. Search for Yigong Shi in:

Competing interests

The authors declare that they have no competing financial interests.

Corresponding author

Correspondence to Yigong Shi.

Supplementary information

Image files

  1. 1.

    Supplementary Figure S1

    Sequence alignment of the human Apaf-1 protein with its homologues in fish, fly and worm (CED-4).

  2. 2.

    Supplementary Figure S2

    Apaf-1 can activate caspase-9 in a 1:1 complex. a, Apaf-1 is capable of binding to caspase-9 in the absence of ATP/dATP.

Word documents

  1. 1.

    Supplementary Table S1

    Diffraction data and refinement statistics.

  2. 2.

    Supplementary Figure Legends

    Legends to accompany Supplementary Figures S1 and S2.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature03465

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.