Crystal structure of baculovirus P35: role of a novel reactive site loop in apoptotic caspase inhibition

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Abstract
The aspartate‐specific caspases are critical protease effectors of programmed cell death and consequently represent important targets for apoptotic intervention. Baculovirus P35 is a potent substrate inhibitor of metazoan caspases, a property that accounts for its unique effectiveness in preventing apoptosis in phylogenetically diverse organisms. Here we report the 2.2 Å resolution crystal structure of P35, the first structure of a protein inhibitor of the death caspases. The P35 monomer possesses a solvent‐exposed loop that projects from the protein's main β‐sheet core and positions the requisite aspartate cleavage site at the loop's apex. Distortion or destabilization of this reactive site loop by site‐directed mutagenesis converted P35 to an efficient substrate which, unlike wild‐type P35, failed to interact stably with the target caspase or block protease activity. Thus, cleavage alone is insufficient for caspase inhibition. These data are consistent with a new model wherein the P35 reactive site loop participates in a unique multi‐step mechanism in which the spatial orientation of the loop with respect to the P35 core determines post‐cleavage association and stoichiometric inhibition of target caspases.