Abstract
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 A 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.
Highlights
Apoptosis or programmed cell death is an active process of cellular self-destruction essential in normal development, tissue homeostasis, and defense against foreign pathogens, including viruses [1, 2]
The structure of wild-type P35 shows a solvent-exposed reactive site loop that projects from the main core of the protein
At the apex of the loop is the caspase recognition site 84-DQMD-87; it is accessible to the target caspase [17]
Summary
Protein Expression and Purification—Baculovirus P35 V71P mutant was cloned into Escherichia coli strain BL21(DE3) using the pET 22(ϩ)b vector expression system (Novagen, Madison, WI), resulting in expression of protein with carboxyl-terminal His-tagged extensions as described previously [16]. There is 1 molecule/ asymmetric unit, giving rise to a solvent content of 62.1% (VM is 3.27 Å3/Da) [21] These crystals diffract to 4.0 Å at home source and 3.1 Å at the synchrotron source. Addition of 1– 8% 2-methyl-2,4-pentanediol to the above-mentioned conditions induced the protein to crystallize in the orthorhombic space group P212121 with unit cell parameters a ϭ 77.6 Å, b ϭ 89.3 Å, and c ϭ 135.1 Å. These crystals appeared as thin needles in 3–5 days and diffracted to 2.7Å resolution at the Stanford Synchrotron Radiation Laboratory. The rotation solutions were applied in a translation search, resulting in a distinct solution with an R-factor of TABLE I Data collection and refinement statistics
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