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Covalent inhibition revealed by the crystal structure of the caspase-8/p35 complex

Abstract

Apoptosis is a highly regulated process that is crucial for normal development and homeostasis of multicellular organisms1,2. The p35 protein from baculoviruses effectively prevents apoptosis by its broad-spectrum caspase inhibition3,4,5,6,7. Here we report the crystal structure of p35 in complex with human caspase-8 at 3.0 Å resolution, and biochemical and mutagenesis studies based on the structural information. The structure reveals that the caspase is inhibited in the active site through a covalent thioester linkage to p35, which we confirmed by gel electrophoresis, hydroxylamine treatment and mass spectrometry experiments. The p35 protein undergoes dramatic conformational changes on cleavage by the caspase. The repositioning of the amino terminus of p35 into the active site of the caspase eliminates solvent accessibility of the catalytic dyad. This may be crucial for preventing hydrolysis of the thioester intermediate, which is supported by the abrogation of inhibitory activity through mutations at the N terminus of p35. The p35 protein also makes conserved contacts with the caspase outside the active-site region, providing the molecular basis for the broad-spectrum inhibitory activity of this protein. We demonstrate a new molecular mechanism of caspase inhibition, as well as protease inhibition in general.

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Figure 1: Structure of the p35/caspase-8 complex.
Figure 2: Biochemical characterization of the p35/caspase-8 complex.
Figure 3: Detailed interaction between p35 and caspase-8.

References

  1. 1

    Thompson, C. B. Apoptosis in the pathogenesis and treatment of disease. Science 267, 1456–1461 (1995).

    ADS  CAS  Article  Google Scholar 

  2. 2

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

    ADS  CAS  Article  Google Scholar 

  3. 3

    Clem, R. J., Fechheimer, M. & Miller, L. K. Prevention of apoptosis by a baculovirus gene during infection of insect cells. Science 254, 1388–1390 (1991).

    ADS  CAS  Article  Google Scholar 

  4. 4

    Bump, N. J. et al. Inhibition of ICE family proteases by baculovirus antiapoptotic protein p35. Science 269, 1885–1888 (1995).

    ADS  CAS  Article  Google Scholar 

  5. 5

    Xue, D. & Horvitz, H. R. Inhibition of the Caenorhabditis elegans cell-death protease CED-3 by a CED-3 cleavage site in baculovirus p35 protein. Nature 377, 248–251 (1995).

    ADS  CAS  Article  Google Scholar 

  6. 6

    Zhou, Q. et al. Interaction of the baculovirus anti-apoptotic protein p35 with caspases. Specificity, kinetics, and characterization of the caspase/p35 complex. Biochemistry 37, 10757–10765 (1998).

    CAS  Article  Google Scholar 

  7. 7

    Ekert, P. G., Silke, J. & Vaux, D. L. Caspase inhibitors. Cell Death Differ. 6, 1081–1086 (1999).

    CAS  Article  Google Scholar 

  8. 8

    Hay, B. A., Wolff, T. & Rubin, G. M. Expression of baculovirus P35 prevents cell death in Drosophila. Development 120, 2121–2129 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    White, K., Tahaoglu, E. & Steller, H. Cell killing by the Drosophila gene reaper. Science 271, 805–807 (1996).

    ADS  CAS  Article  Google Scholar 

  10. 10

    Beidler, D. R., Tewari, M., Friesen, P. D., Poirier, G. & Dixit, V. M. The baculovirus p35 protein inhibits Fas- and tumor necrosis factor-induced apoptosis. J. Biol. Chem. 270, 16526–16528 (1995).

    CAS  Article  Google Scholar 

  11. 11

    Robertson, N. M. et al. Baculovirus P35 inhibits the glucocorticoid-mediated pathway of cell death. Cancer Res. 57, 43–47 (1997).

    CAS  PubMed  Google Scholar 

  12. 12

    Hisahara, S. et al. Targeted expression of baculovirus p35 caspase inhibitor in oligodendrocytes protects mice against autoimmune-mediated demyelination. EMBO J. 19, 341–348 (2000).

    CAS  Article  Google Scholar 

  13. 13

    Fisher, A. J., Cruz, W., Zoog, S. J., Schneider, C. L. & Friesen, P. D. Crystal structure of baculovirus P35: role of a novel reactive site loop in apoptotic caspase inhibition. EMBO J. 18, 2031–2039 (1999).

    CAS  Article  Google Scholar 

  14. 14

    Zoog, S. J., Bertin, J. & Friesen, P. D. Caspase inhibition by baculovirus P35 requires interaction between the reactive site loop and the beta-sheet core. J. Biol. Chem. 274, 25995–26002 (1999).

    CAS  Article  Google Scholar 

  15. 15

    Sambrook, J., Fritsch, E. F. & Maniatis, T. Molecular Cloning, 2nd edn (Cold Spring Harbor Laboratory Press, New York, 1989).

    Google Scholar 

  16. 16

    Bruice, T. C. & Benkovic, S. J. Bioorganic Mechanisms (Benjamin, New York, 1966).

    Google Scholar 

  17. 17

    Owen, W. G., Penick, G. D., Yoder, E. & Poole, B. L. Evidence for an ester bond between thrombin and heparin cofactor. Thromb. Haemost. 35, 87–95 (1976).

    CAS  Article  Google Scholar 

  18. 18

    Stennicke, H. R. & Salvesen, G. S. Catalytic properties of the caspases. Cell Death Differ. 6, 1054–1059 (1999).

    CAS  Article  Google Scholar 

  19. 19

    Watt, W. et al. The atomic-resolution structure of human caspase-8, a key activator of apoptosis. Structure Fold Des. 7, 1135–1143 (1999).

    CAS  Article  Google Scholar 

  20. 20

    Blanchard, H. et al. The three-dimensional structure of caspase-8: an initiator enzyme in apoptosis. Structure Fold Des. 7, 1125–1133 (1999).

    CAS  Article  Google Scholar 

  21. 21

    Bertin, J. et al. Apoptotic suppression by baculovirus P35 involves cleavage by and inhibition of a virus-induced CED-3/ICE-like protease. J. Virol. 70, 6251–6259 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Bode, W. & Huber, R. Structural basis of the endoproteinase–protein inhibitor interaction. Biochim. Biophys. Acta 1477, 241–252 (2000).

    CAS  Article  Google Scholar 

  23. 23

    Wright, H. T. & Scarsdale, J. N. Structural basis for serpin inhibitor activity. Proteins 22, 210–225 (1995).

    CAS  Article  Google Scholar 

  24. 24

    Huntington, J. A., Read, R. J. & Carrell, R. W. Structure of a serpin–protease complex shows inhibition by deformation. Nature 407, 923–926 (2000).

    ADS  CAS  Article  Google Scholar 

  25. 25

    Tong, L. REPLACE, a suite of computer programs for molecular-replacement calculations. J. Appl. Cryst. 26, 748–751 (1993).

    Article  Google Scholar 

  26. 26

    Jones, T. A., Zou, J. -Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building models in electron density maps and the location of errors in those models. Acta Crystallogr. A 47, 110–119 (1991).

    Article  Google Scholar 

  27. 27

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

    CAS  Article  Google Scholar 

  28. 28

    Park, Y. C. et al. A novel mechanism of TRAF signaling revealed by structural and functional analyses of the TRADD–TRAF2 interaction. Cell 101, 777–787 (2000).

    CAS  Article  Google Scholar 

  29. 29

    Myszka, D. G. Improving biosensor analysis. Mol. Recogn. 12, 1–6 (1999).

    Article  Google Scholar 

  30. 30

    Myszka, D. G. & Morton, T. A. CLAMP: a biosensor kinetic data analysis program. Trends Biochem. Sci. 23, 149–150 (1998).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank K. D'Amico for access to the COM-CAT beamline at the APS; C. Ogata for access to the X4A beamline at NSLS; L. Tong for help with data collection and for critical reading of the manuscript; J. Kerwin for mass spectrometry experiments; J. Luft and G. DeTitta for initial screening of crystallization conditions for a p35/caspase-3 complex, which served as leads for the crystallization of the p35/caspase-8 complex; and C. Lima, T. Muir, B. Chait, G. Dodson, H. T. Wright and T. Begley for insightful discussions. This work was supported by the Speaker's Fund for Biomedical Research and the departmental startup fund. H.W. is a Pew Scholar in the Biomedical Sciences.

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Correspondence to Hao Wu.

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Xu, G., Cirilli, M., Huang, Y. et al. Covalent inhibition revealed by the crystal structure of the caspase-8/p35 complex. Nature 410, 494–497 (2001). https://doi.org/10.1038/35068604

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