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 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.
dela Cruz
W P
WP
Department of Chemistry, University of California, Davis, California 95616, USA.
Friesen
P D
PD
Fisher
A J
AJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017209:Apoptosis / Q000502:physiology; D001224:Aspartic Acid; D016367:Baculoviridae; D001665:Binding Sites; D020169:Caspases / Q000378:metabolism*; D018360:Crystallography, X-Ray; D051034:Inhibitor of Apoptosis Proteins; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017924:Nucleopolyhedroviruses; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
D051034:Inhibitor of Apoptosis Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C086430:inhibitor of apoptosis, Nucleopolyhedrovirus; D001224:Aspartic Acid; D020169:Caspases