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Published on: March 5, 2018
Mutational analyses of the p35-caspase interaction. A bowstring kinetic model of caspase inhibition by p35
Guozhou Xu1, Rebecca L Rich, Clemens Steegborn
1Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021, USA.
Abstract:
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.
Insights
Baculoviral p35 protein inhibits apoptosis by blocking caspases. Structural analysis reveals key elements like the reactive site loop and N-terminal cysteine are crucial for p35
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Apoptosis, or programmed cell death, is a critical biological process.
- Caspases are key intracellular cysteine proteases that execute apoptosis.
- Baculoviral p35 protein is a potent inhibitor of caspases, broadly blocking apoptosis.
Purpose of the Study:
- To investigate the structural mechanisms underlying p35's caspase inhibition.
- To identify critical structural elements involved in pre- and post-cleavage p35/caspase interactions.
- To elucidate the kinetic model governing p35's function.
Main Methods:
- Surface plasmon resonance (SPR) biosensor measurements.
- Pull-down assays.
- Kinetic inhibition experiments.
- Mutational analyses of p35 and caspase interactions.
Main Results:
- Identified a strict requirement for an N-terminal cysteine in p35 for effective caspase inhibition.
- Demonstrated the importance of the rigidity of the reactive site loop (RSL) in p35.
- Revealed that p35 forms a stable thioester bond with the caspase active site cysteine.
- Proposed a bowstring kinetic model where pre-cleavage interaction tension drives post-cleavage inhibition.
Conclusions:
- p35 utilizes a unique mechanism involving covalent bond formation and conformational changes for caspase inhibition.
- Specific structural features of p35, including the N-terminal cysteine and RSL, are essential for its inhibitory function.
- The bowstring kinetic model provides a framework for understanding the dynamics of p35-caspase interactions.
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