Crystal structure of baculovirus P35 reveals a novel conformational change in the reactive site loop after caspase cleavage

Baculovirus P35 is a universal suppressor of apoptosis that stoichiometrically inhibits cellular caspases in a novel cleavage-dependent mechanism. Upon caspase cleavage at Asp-87, the 10- and 25-kDa cleavage products of P35 remain tightly associated with the inhibited caspase. Mutations in the alpha -helix of the reactive site loop preceding the cleavage site abrogate caspase inhibition and antiapoptotic activity. Substitution of Pro for Val-71, which is located in the middle of this alpha -helix, produces a protein that is cleaved at the requisite Asp-87 but does not remain bound to the caspase. This loss-of-function mutation provided the opportunity to structurally analyze the conformational changes of the P35 reactive site loop after caspase cleavage. We report here the 2.7 Angstrom resolution crystal structure of V71P-mutated P35 after cleavage by human caspase-3. The structure reveals a large movement in the carboxyl-terminal side of the reactive site loop that swings down and forms a new beta -strand that augments an existing beta -sheet. Additionally, the hydrophobic amino terminus releases and extends away from the protein core. Similar movements occur when P35 forms an inhibitory complex with human caspase-8. These findings suggest that the alpha -helix mutation may alter the sequential steps or kinetics of the conformational changes required for inhibition, thereby causing P35 loss of function.