Mutational analyses of the p35-caspase interaction. A bowstring kinetic model of caspase inhibition by p35.
Apoptosis is a highly regulated multistep process for programmed cellular destruction. It is centered on the activation of a group of intracellular cysteine proteases known as caspases. The baculoviral p35 protein effectively blocks apoptosis through its broad spectrum caspase inhibition. It harbors a caspase recognition sequence within a highly protruding reactive site loop (RSL), which gets cleaved by a target caspase before the formation of a tight complex. The crystal structure of the post-cleavage complex between p35 and caspase-8 shows that p35 forms a thioester bond with the active site cysteine of the caspase. The covalent bond is prevented from hydrolysis by the N terminus of p35, which repositions into the active site of the caspase to eliminate solvent accessibility of the catalytic residues. Here, we report mutational analyses of the pre-cleavage and post-cleavage p35/caspase interactions using surface plasmon resonance biosensor measurements, pull-down assays and kinetic inhibition experiments. The experiments identify important structural elements for caspase inhibition by p35, including the strict requirement for a Cys at the N terminus of p35 and the rigidity of the RSL. A bowstring kinetic model for p35 function is derived in which the tension generated in the bowstring system during the pre-cleavage interaction is crucial for the fast post-cleavage conformational changes required for inhibition.
Xu
Guozhou
G
Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021, USA.
Rich
Rebecca L
RL
Steegborn
Clemens
C
Min
Tongpil
T
Huang
Yihua
Y
Myszka
David G
DG
Wu
Hao
H
D017209:Apoptosis / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000235:genetics*; D004252:DNA Mutational Analysis; D004791:Enzyme Inhibitors / Q000378:metabolism*; D006801:Humans; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D013379:Substrate Specificity; D014764:Viral Proteins
D001425:Bacterial Outer Membrane Proteins; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D008074:Lipoproteins; D014764:Viral Proteins; C492234:p35 protein, Baculovirus; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases