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.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Steller, H. Mechanisms and genes of cellular suicide. Science 267 , 1445–1449 (1995).
Jacobson, M. D., Weil, M. & Raff, M. C. Programmed cell death in animal development. Cell 88, 347–354 ( 1997).
Hengartner, M. O. Programmed cell death in invertebrates. Curr. Opin. Genet. Dev. 6, 34–38 (1996 ).
Horvitz, H. R. Genetic control of programmed cell death in the nematode Caenorhabditis elegans. Cancer Res. 59, 1701– 1706 (1999).
Thompson, C. B. Apoptosis in the pathogenesis and treatment of disease. Science 267, 1456–1462 ( 1995).
Green, D. R. & Martin, S. J. The killer and the executioner: how apoptosis controls malignancy. Curr. Opin. Immunol. 7, 694–703 (1995).
Thornberry, N. A. & Lazebnik, Y. Caspases: Enemies within. Science 281, 1312– 1316 (1998).
Chinnaiyan, A. M. & Dixit, V. M. The cell-death machine. Curr. Biol. 6, 555– 562 (1996).
Deveraux, Q. L. & Reed, J. C. IAP family proteins—suppressors of apoptosis. Genes Dev. 13, 239– 252 (1999).
Miller, L. K. An exegesis of IAPs: salvation and surprises from BIR motifs. Trends Cell Biol. 9, 323–328 (1999).
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).
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).
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).
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).
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).
Yang, X., Chang, H. Y. & Baltimore, D. Essential role of CED-4 oligomerization in CED-3 activation and apoptosis. Science 281, 1355– 1357 (1998).
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).
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).
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).
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).
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).
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).
Takahashi, R. et al. A single BIR domain of XIAP sufficient for inhibiting caspases. J. Biol. Chem. 273, 7787– 7790 (1998).
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).
Enari, M. et al. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391, 43– 50 (1998).
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).
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).
Rotonda, J. et al. The three-dimensional structure of apopain/CPP32, a key mediator of apoptosis. Nature Struct. Biol. 3, 619 –625 (1996).
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).
Sun, C. et al. NMR structure and mutagenesis of the inhibitor-of-apoptosis protein XIAP. Nature 401, 818–822 (1999).
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).
Terwilliger, T. C. & Berendzen, J. Correlated phasing of multiple isomorphous replacement data. Acta Crystallogr. D 52, 749–757 (1996).
Collaborative Computational Project, N. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 ( 1994).
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).
Brunger, A. T. et al. Crystallography and NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).
Klaulis, P. J. Molscript: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946 –950 (1991).
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).
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.
About this article
Cite this article
Chai, J., Du, C., Wu, JW. et al. Structural and biochemical basis of apoptotic activation by Smac/DIABLO . Nature 406, 855–862 (2000). https://doi.org/10.1038/35022514
Skd3 (human ClpB) is a potent mitochondrial protein disaggregase that is inactivated by 3-methylglutaconic aciduria-linked mutations
Rice Bran Phenolic Extract Confers Protective Effects against Alcoholic Liver Disease in Mice by Alleviating Mitochondrial Dysfunction via the PGC-1α-TFAM Pathway Mediated by microRNA-494-3p
Journal of Agricultural and Food Chemistry (2020)
Recent advances in the development of protein–protein interactions modulators: mechanisms and clinical trials
Signal Transduction and Targeted Therapy (2020)
Monoglyceride lipase mediates tumor-suppressive effects by promoting degradation of X-linked inhibitor of apoptosis protein
Cell Death & Differentiation (2020)