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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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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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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