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Structure of the CED-4–CED-9 complex provides insights into programmed cell death in Caenorhabditis elegans

Nature volume 437, pages 831–837 (2005)Cite this article

Abstract

Interplay among four genes—egl-1, ced-9, ced-4 and ced-3—controls the onset of programmed cell death in the nematode Caenorhabditis elegans. Activation of the cell-killing protease CED-3 requires CED-4. However, CED-4 is constitutively inhibited by CED-9 until its release by EGL-1. Here we report the crystal structure of the CED-4–CED-9 complex at 2.6 Å resolution, and a complete reconstitution of the CED-3 activation pathway using homogeneous proteins of CED-4, CED-9 and EGL-1. One molecule of CED-9 binds to an asymmetric dimer of CED-4, but specifically recognizes only one of the two CED-4 molecules. This specific interaction prevents CED-4 from activating CED-3. EGL-1 binding induces pronounced conformational changes in CED-9 that result in the dissociation of the CED-4 dimer from CED-9. The released CED-4 dimer further dimerizes to form a tetramer, which facilitates the autoactivation of CED-3. Together, our studies provide important insights into the regulation of cell death activation in C. elegans.

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Figure 1: Overall structure of the CED-4–CED-9 complex.
Figure 2: Mechanisms of CED-4 recognition by CED-9, and CED-4 release by EGL-1.
Figure 3: Structural analyses of the CED-4a–CED-4b interface and ATP binding.
Figure 4: Free CED-4 forms a tetramer.
Figure 5: Complete reconstitution of the CED-3 activation pathway using homogeneous proteins of CED-4, CED-9 and EGL-1.

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References

  1. Horvitz, H. R. Worms, Life, and Death (Nobel Lecture). ChemBioChem 4, 697–711 (2003)

    Article  CAS  PubMed  Google Scholar 

  2. Horvitz, H. R. Genetic control of programmed cell death in the nematode Caenorhabditis elegans. Cancer Res. 59, 1701–1706 (1999)

    Google Scholar 

  3. Yuan, J., Shaham, S., Ledoux, S., Ellis, H. M. & Horvitz, H. R. The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1β-converting enzyme. Cell 75, 641–652 (1993)

    Article  CAS  PubMed  Google Scholar 

  4. Xue, D., Shaham, S. & Horvitz, H. R. The Caenorhabditis elegans cell-death protein CED-3 is a cysteine protease with substrate specificities similar to those of the human CPP32 protease. Genes Dev. 10, 1073–1083 (1996)

    Article  CAS  PubMed  Google Scholar 

  5. Thornberry, N. A. & Lazebnik, Y. Caspases: Enemies within. Science 281, 1312–1316 (1998)

    Article  CAS  PubMed  Google Scholar 

  6. Yuan, J. & Horvitz, H. R. The Caenorhabditis elegans cell death gene ced-4 encodes a novel protein and is expressed during the period of extensive programmed cell death. Development 116, 309–320 (1992)

    CAS  PubMed  Google Scholar 

  7. Chinnaiyan, A. M., O'Rourke, K., Lane, B. R. & Dixit, V. M. Interaction of CED-4 with CED-3 and CED-9: a molecular framework for cell death. Science 275, 1122–1126 (1997)

    Article  CAS  PubMed  Google Scholar 

  8. Irmler, M., Hofmann, K., Vaux, D. & Tschopp, J. Direct physical interaction between the Caenorhabditis elegans ‘death proteins’ CED-3 and CED-4. FEBS Lett. 406, 189–190 (1997)

    Article  CAS  PubMed  Google Scholar 

  9. Seshagiri, S. & Miller, L. K. Caenorhabditis elegans CED-4 stimulates CED-3 processing and CED-3-induced apoptosis. Curr. Biol. 7, 455–460 (1997)

    Article  CAS  PubMed  Google Scholar 

  10. Wu, D., Wallen, H. D. & Nunez, G. Interaction and regulation of subcellular localization of CED-4 by CED-9. Science 275, 1126–1129 (1997)

    Article  CAS  PubMed  Google Scholar 

  11. Yang, X., Chang, H. Y. & Baltimore, D. Essential role of CED-4 oligomerization in CED-3 activation and apoptosis. Science 281, 1355–1357 (1998)

    Article  ADS  CAS  PubMed  Google Scholar 

  12. Hengartner, M. O. & Horvitz, H. R. C. elegans cell survival gene ced-9 encodes a functional homolog of the mammalian proto-oncogene bcl-2. Cell 76, 665–676 (1994)

    Article  CAS  PubMed  Google Scholar 

  13. Chen, F. et al. Translocation of C. elegans CED-4 to nuclear membranes during programmed cell death. Science 287, 1485–1489 (2000)

    Article  ADS  CAS  PubMed  Google Scholar 

  14. James, C., Gschmeissner, S., Fraser, A. & Evan, G. I. CED-4 induces chromatin condensation in Schizosaccharomyces pombe and is inhibited by direct physical association with CED-9. Curr. Biol. 7, 246–252 (1997)

    Article  CAS  PubMed  Google Scholar 

  15. Spector, M. S., Desnoyers, S., Hoeppner, D. J. & Hengartner, M. O. Interaction between the C. elegans cell-death regulators CED-9 and CED-4. Nature 385, 653–656 (1997)

    Article  ADS  CAS  PubMed  Google Scholar 

  16. Conradt, B. & Horvitz, H. R. The C. elegans protein EGL-1 is required for programmed cell death and interacts with the Bcl-2-like protein CED-9. Cell 93, 519–529 (1998)

    Article  CAS  PubMed  Google Scholar 

  17. del Peso, L., Gonzalez, V. M. & Nunez, G. Caenorhabditis elegans EGL-1 disrupts the interaction of CED-9 with CED-4 and promotes CED-3 activation. J. Biol. Chem. 273, 33495–33500 (1998)

    Article  CAS  PubMed  Google Scholar 

  18. del Peso, L., Gonzalez, V. M., Inohara, N., Ellis, R. E. & Nunez, G. Disruption of the CED-9·CED-4 complex by EGL-1 is a critical step for programmed cell death in Caenorhabditis elegans. J. Biol. Chem. 275, 27205–27211 (2000)

    CAS  PubMed  Google Scholar 

  19. Parrish, J., Metters, H., Chen, L. & Xue, D. Demonstration of the in vivo interaction of key cell death regulators by structure-based design of second-site suppressors. Proc. Natl Acad. Sci. USA 97, 11916–11921 (2000)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  20. Yan, N. et al. Structural, biochemical, and functional analyses of CED-9 recognition by the proapoptotic proteins EGL-1 and CED-4. Mol. Cell 15, 999–1006 (2004)

    Article  CAS  PubMed  Google Scholar 

  21. Riedl, S. J., Li, W., Chao, Y., Schwarzenbacher, R. & Shi, Y. Structure of the apoptotic protease activating factor 1 bound to ADP. Nature 434, 926–933 (2005)

    Article  ADS  CAS  PubMed  Google Scholar 

  22. Zou, H., Henzel, W. J., Liu, X., Lutschg, A. & Wang, X. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90, 405–413 (1997)

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  24. Liu, J. et al. Structure and function of Cdc6/Cdc18: implications for origin recognition and checkpoint control. Mol. Cell 6, 637–648 (2000)

    Article  CAS  PubMed  Google Scholar 

  25. Singleton, M. R. et al. Conformational changes induced by nucleotide binding in Cdc6/ORC from Aeropyrum pernix. J. Mol. Biol. 343, 547–557 (2004)

    Article  CAS  PubMed  Google Scholar 

  26. Lupas, A. N. & Martin, J. AAA proteins. Curr. Opin. Struct. Biol. 12, 746–753 (2002)

    Article  CAS  PubMed  Google Scholar 

  27. Jaroszewski, L., Rychlewski, L., Reed, J. C. & Godzik, A. ATP-activated oligomerization as a mechanism for apoptosis regulation: fold and mechanism prediction for CED-4. Proteins 39, 197–203 (2000)

    Article  CAS  PubMed  Google Scholar 

  28. Woo, J. S. et al. Unique structural features of a BCL-2 family protein CED-9 and biophysical characterization of CED-9/EGL-1 interactions. Cell Death Differ. 10, 1310–1325 (2003)

    Article  CAS  PubMed  Google Scholar 

  29. Hugunin, M., Quintal, L. J., Mankovich, J. A. & Ghayur, T. Protease activity of in vitro transcribed and translated Caenorhabditis elegans cell death gene (ced-3) product. J. Biol. Chem. 271, 3517–3522 (1996)

    Article  CAS  PubMed  Google Scholar 

  30. Shaham, S. & Horvitz, H. R. An alternatively spliced C. elegans ced-4 RNA encodes a novel cell death inhibitor. Cell 86, 201–208 (1996)

    Article  CAS  PubMed  Google Scholar 

  31. Ottilie, S. et al. Mutational analysis of the interacting cell death regulators CED-9 and CED-4. Cell Death Differ. 4, 526–533 (1997)

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  33. Uson, I. & Sheldrick, G. M. Advance in direct methods for protein crystallography. Curr. Opin. Struct. Biol. 9, 643–648 (1999)

    Article  CAS  PubMed  Google Scholar 

  34. Hao, Q. ABS: A program to determine absolute configuration and evaluate anomalous scatter substructure. J. Appl. Crystallogr. 37, 498–499 (2004)

    Article  CAS  Google Scholar 

  35. Terwilliger, T. C. & Berendzen, J. Automated structure solution for MIR and MAD. Acta Crystallogr. D 55, 849–861 (1999)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Abrahams, J. P. & Leslie, A. G. Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Crystallogr. D 52, 30–42 (1996)

    Article  CAS  PubMed  Google Scholar 

  37. Jones, T. A., Zou, J.-Y., Cowan, S. W. & Kjeldgaard, M. 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)

    Article  PubMed  Google Scholar 

  38. Brunger, A. T. et al. Crystallography and NMR System: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  CAS  PubMed  Google Scholar 

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

    Article  Google Scholar 

  40. Nicholls, A., Sharp, K. A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins Struct. Funct. Genet. 11, 281–296 (1991)

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank H. R. Horvitz for complementary DNA sequences of egl-1, ced-9, ced-4 and ced-3, and for his encouragement. We thank V. Dixit and X. Yang for providing ced-3 constructs, and M. Becker and A. Saxena for beamtime at NSLS. This research was supported by NIH grants to Y.S. and D.X. H.L. acknowledges support from the Brookhaven National Laboratory LDRD program and a Department of Energy grant. The atomic coordinates of the CED-4–CED-9 complex have been deposited in the Protein Data Bank with the accession number 2A5Y. Author Contributions N.Y. performed the bulk of the experiments. N.Y. and Y.S. designed and interpreted the bulk of the experiments. J.C., E.S.L., L.G., Q.L., J.H., J.-W.W. and D.K. contributed to experiments. J.C., E.S.L., L.G., Q.L., J.H., J.-W.W., D.K., H.L., Q.H. and D.X. contributed to data analysis and interpretation. N.Y. and Y.S. wrote the paper. J.C. and E.S.L. contributed equally to this work.

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Authors and Affiliations

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

    Nieng Yan, Jijie Chai, Lichuan Gu, Jia-Wei Wu & Yigong Shi

  2. Department of Molecular, Cellular and Developmental Biology, University of Colorado, Colorado, 80309, Boulder, USA

    Eui Seung Lee, David Kokel & Ding Xue

  3. Department of Life Science, Gwangju Institute of Science and Technology, 500-712, Gwangju, Republic of Korea

    Eui Seung Lee

  4. Molecular Biology and Genetics, Cornell University, New York, 14853, Ithaca, USA

    Qun Liu & Quan Hao

  5. Biology Department, Brookhaven National Laboratory, New York, 11973, Upton, USA

    Jiaqing He & Huilin Li

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Supplementary information

Supplementary Table 1

Summary of Crystallographic Analysis. (DOC 24 kb)

Supplementary Table 2

Biological and biochemical characterization of CED-4 mutants. (DOC 48 kb)

Supplementary Figure 1

Mapping of the surface features onto the primary sequences of CED-4 (JPG 448 kb)

Supplementary Figure 2

Mechanism of CED-4 release induced by EGL-1 binding. (JPG 266 kb)

Supplementary Figure 3

CED-4a and CED-4b are structurally similar. (JPG 514 kb)

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Yan, N., Chai, J., Lee, E. et al. Structure of the CED-4–CED-9 complex provides insights into programmed cell death in Caenorhabditis elegans. Nature 437, 831–837 (2005). https://doi.org/10.1038/nature04002

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