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

The aspartate-specific caspases are critical protease effecters of programm ed cell death and consequently represent important targets for apoptotic in tervention. Baculovirus P35 is a potent substrate inhibitor of metazoan cas pases, a property that accounts for its unique effectiveness in preventing apoptosis in phylogenetically diverse organisms. Here we report the 2.2 Ang strom resolution crystal structure of P35, the first structure of a protein inhibitor of the death caspases. The P35 monomer possesses a solvent-expos ed loop that projects from the protein's main beta-sheet core and positions the requisite aspartate cleavage site at the loop's apex. Distortion or de stabilization of this reactive site loop by site-directed mutagenesis conve rted P35 to an efficient substrate which, unlike wild-type P35, failed to i nteract stably with the target caspase or block protease activity. Thus, cl eavage alone is insufficient for caspase inhibition, These data are consist ent with a new model wherein the P35 reactive site loop participates in a u nique multi-step mechanism in which the spatial orientation of the loop wit h respect to the P35 core determines post-cleavage association and stoichio metric inhibition of target caspases.