TY - JOUR
T1 - Crystal Structure of Baculovirus P35 Reveals a Novel Conformational Change in the Reactive Site Loop after Caspase Cleavage
AU - Dela Cruz, Wilfred P.
AU - Friesen, Paul D.
AU - Fisher, Andrew J
PY - 2001/8/31
Y1 - 2001/8/31
N2 - 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 α-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 α-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 Å 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 β-strand that augments an existing β-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 α-helix mutation may alter the sequential steps or kinetics of the conformational changes required for inhibition, thereby causing P35 loss of function.
AB - 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 α-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 α-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 Å 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 β-strand that augments an existing β-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 α-helix mutation may alter the sequential steps or kinetics of the conformational changes required for inhibition, thereby causing P35 loss of function.
UR - http://www.scopus.com/inward/record.url?scp=0035980091&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0035980091&partnerID=8YFLogxK
U2 - 10.1074/jbc.M103930200
DO - 10.1074/jbc.M103930200
M3 - Article
C2 - 11402050
AN - SCOPUS:0035980091
VL - 276
SP - 32933
EP - 32939
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 35
ER -