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

Guozhou Xu, Maurizio Cirilli, Yihua Huang, Rebecca L. Rich, David G. Myszka and Hao Wu ()
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Guozhou Xu: Weill Medical College of Cornell University and
Maurizio Cirilli: Weill Medical College of Cornell University and
Yihua Huang: Graduate School of Medical Sciences of Cornell University
Rebecca L. Rich: Center for Biomolecular Interaction Analysis, School of Medicine, University of Utah
David G. Myszka: Center for Biomolecular Interaction Analysis, School of Medicine, University of Utah
Hao Wu: Weill Medical College of Cornell University and

Nature, 2001, vol. 410, issue 6827, 494-497

Abstract: 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.

Date: 2001
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DOI: 10.1038/35068604

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