Structural and biochemical basis of apoptotic activation by Smac/DIABLO


Apoptosis (programmed cell death), an essential process in the development and homeostasis of metazoans, is carried out by caspases. The mitochondrial protein Smac/DIABLO performs a critical function in apoptosis by eliminating the inhibitory effect of IAPs (inhibitor of apoptosis proteins) on caspases. Here we show that Smac/DIABLO promotes not only the proteolytic activation of procaspase-3 but also the enzymatic activity of mature caspase-3, both of which depend upon its ability to interact physically with IAPs. The crystal structure of Smac/DIABLO at 2.2 Å resolution reveals that it homodimerizes through an extensive hydrophobic interface. Missense mutations inactivating this dimeric interface significantly compromise the function of Smac/DIABLO. As in the Drosophila proteins Reaper, Grim and Hid, the amino-terminal amino acids of Smac/DIABLO are indispensable for its function, and a seven-residue peptide derived from the amino terminus promotes procaspase-3 activation in vitro. These results establish an evolutionarily conserved structural and biochemical basis for the activation of apoptosis by Smac/DIABLO.

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Figure 1: Schematic representation of Smac structure.
Figure 2: Specificity at the dimeric interface.
Figure 3: Functional significance of the dimeric interface in Smac.
Figure 4: Functional significance of the N terminus of Smac.
Figure 5: N-terminal peptides of Smac directly promote the activation of procaspase-3.
Figure 6: Model of Smac function.


  1. 1

    Steller, H. Mechanisms and genes of cellular suicide. Science 267 , 1445–1449 (1995).

    ADS  CAS  Article  Google Scholar 

  2. 2

    Jacobson, M. D., Weil, M. & Raff, M. C. Programmed cell death in animal development. Cell 88, 347–354 ( 1997).

    CAS  Article  Google Scholar 

  3. 3

    Hengartner, M. O. Programmed cell death in invertebrates. Curr. Opin. Genet. Dev. 6, 34–38 (1996 ).

    CAS  Article  Google Scholar 

  4. 4

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

    Google Scholar 

  5. 5

    Thompson, C. B. Apoptosis in the pathogenesis and treatment of disease. Science 267, 1456–1462 ( 1995).

    ADS  CAS  Article  Google Scholar 

  6. 6

    Green, D. R. & Martin, S. J. The killer and the executioner: how apoptosis controls malignancy. Curr. Opin. Immunol. 7, 694–703 (1995).

    CAS  Article  Google Scholar 

  7. 7

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

    CAS  Article  Google Scholar 

  8. 8

    Chinnaiyan, A. M. & Dixit, V. M. The cell-death machine. Curr. Biol. 6, 555– 562 (1996).

    CAS  Article  Google Scholar 

  9. 9

    Deveraux, Q. L. & Reed, J. C. IAP family proteins—suppressors of apoptosis. Genes Dev. 13, 239– 252 (1999).

    CAS  Article  Google Scholar 

  10. 10

    Miller, L. K. An exegesis of IAPs: salvation and surprises from BIR motifs. Trends Cell Biol. 9, 323–328 (1999).

    CAS  Article  Google Scholar 

  11. 11

    Wang, S., Hawkins, C., Yoo, S., Muller, H.-A. & Hay, B. The Drosophila caspase inhibitor DIAP1 is essential for cell survival and is negatively regulated by HID. Cell 98, 453–463 (1999).

    CAS  Article  Google Scholar 

  12. 12

    Goyal, L., McCall, K., Agapite, J., Hartwieg, E. & Steller, H. Induction of apoptosis by Drosophila reaper, hid and grim through inhibition of IAP function. EMBO J. 19, 589–597 (2000).

    CAS  Article  Google Scholar 

  13. 13

    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).

    CAS  Article  Google Scholar 

  14. 14

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

    CAS  Article  Google Scholar 

  15. 15

    Srinivasula, S. M., Ahmad, M., Fernandes-Alnemri, T. & Alnemri, E. S. Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol. Cell 1, 949–957 ( 1998).

    CAS  Article  Google Scholar 

  16. 16

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

    ADS  CAS  Article  Google Scholar 

  17. 17

    Hu, Y., Ding, L., Spencer, D. M. & Nunez, G. WD-40 repeat region regulates Apaf-1 self-association and procaspase-9 activation. J. Biol. Chem. 273, 33489–33494 (1998).

    CAS  Article  Google Scholar 

  18. 18

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

    CAS  Article  Google Scholar 

  19. 19

    Saleh, A., Srinivasula, S. M., Acharya, S., Fishel, R. & Alnemri, E. S. Cytochrome c and dATP-mediated oligomerization of Apaf-1 is a prerequisite for procaspase-9 activation. J. Biol. Chem. 274, 17941–17945 (1999).

    CAS  Article  Google Scholar 

  20. 20

    Du, C., Fang, M., Li, Y., Li, L. & Wang, X. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 102, 33–42 (2000).

    CAS  Article  Google Scholar 

  21. 21

    Verhagen, A. et al. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing inhibitor of apoptosis (IAP) proteins. Cell 102, 43–53 (2000).

    CAS  Article  Google Scholar 

  22. 22

    Deveraux, Q. L. et al. Cleavage of human inhibitor of apoptosis protein XIAP results in fragments with distinct specificities for caspases. EMBO J. 18, 5242–5251 ( 1999).

    CAS  Article  Google Scholar 

  23. 23

    Takahashi, R. et al. A single BIR domain of XIAP sufficient for inhibiting caspases. J. Biol. Chem. 273, 7787– 7790 (1998).

    CAS  Article  Google Scholar 

  24. 24

    Liu, X., Zou, H., Slaughter, C. & Wang, X. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 89, 175– 184 (1997).

    CAS  Article  Google Scholar 

  25. 25

    Enari, M. et al. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391, 43– 50 (1998).

    ADS  CAS  Article  Google Scholar 

  26. 26

    Liu, X. et al. The 40-kDa subunit of DNA fragmentation factor induces DNA fragmentation and chromatin condensation during apoptosis. Proc. Natl Acad. Sci. USA 95, 8461–8466 ( 1998).

    ADS  CAS  Article  Google Scholar 

  27. 27

    Hirel, P.-H., Schmitter, J.-M., Dessen, P., Fayat, G. & Blanquet, S. Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid. Proc. Natl Acad. Sci. USA 86, 8247–8251 ( 1989).

    ADS  CAS  Article  Google Scholar 

  28. 28

    Rotonda, J. et al. The three-dimensional structure of apopain/CPP32, a key mediator of apoptosis. Nature Struct. Biol. 3, 619 –625 (1996).

    CAS  Article  Google Scholar 

  29. 29

    Hozak, R. R., Manji, G. A. & Friesen, P. D. The BIR motifs mediate dominant interference and oligomerization of inhibitor of apoptosis Op-IAP. Mol. Cell. Biol. 20, 1877–1885 ( 2000).

    CAS  Article  Google Scholar 

  30. 30

    Sun, C. et al. NMR structure and mutagenesis of the inhibitor-of-apoptosis protein XIAP. Nature 401, 818–822 (1999).

    ADS  CAS  Article  Google Scholar 

  31. 31

    Liu, X., Kim, C. N., Yang, J., Jemmerson, R. & Wang, X. Induction of apoptosis program in cell-free extracts: Requirement for dATP and cytochrome c. Cell 86, 147–157 (1996).

    CAS  Article  Google Scholar 

  32. 32

    Terwilliger, T. C. & Berendzen, J. Correlated phasing of multiple isomorphous replacement data. Acta Crystallogr. D 52, 749–757 (1996).

    CAS  Article  Google Scholar 

  33. 33

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

    Article  Google Scholar 

  34. 34

    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  Google Scholar 

  35. 35

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

    CAS  Article  Google Scholar 

  36. 36

    Klaulis, 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 

  37. 37

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

    CAS  Article  Google Scholar 

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We thank D. Vaux for providing XIAP cDNA; E. Alnemri for providing caspase-3 expression vector; F. Hughson for critically reading the manuscript; and N. Hunt for secretarial assistance. This work was supported by start-up funds from Princeton University (to Y.S.) and Howard Hughes Medical Institute (to X.W.). Y.S. is a Searle Scholar and a Rita Allen Scholar.

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Correspondence to Yigong Shi.

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Chai, J., Du, C., Wu, JW. et al. Structural and biochemical basis of apoptotic activation by Smac/DIABLO . Nature 406, 855–862 (2000).

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