Covalent inhibition revealed by the crystal structure of the caspase-8/p35 complex.
Apoptosis is a highly regulated process that is crucial for normal development and homeostasis of multicellular organisms. The p35 protein from baculoviruses effectively prevents apoptosis by its broad-spectrum caspase inhibition. Here we report the crystal structure of p35 in complex with human caspase-8 at 3.0 A 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.
Xu
G
G
Department of Biochemistry, Weill Medical College of Cornell University, New York, New York, 10021 USA
Cirilli
M
M
Huang
Y
Y
Rich
R L
RL
Myszka
D G
DG
Wu
H
H
D001665:Binding Sites; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology
D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D014764:Viral Proteins; C081618:p35 protein, Nucleopolyhedrovirus; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases