Reactive-site cleavage residues confer target specificity to baculovirus P49, a dimeric member of the P35 family of caspase inhibitors.
Baculovirus proteins P49 and P35 are potent suppressors of apoptosis in diverse organisms. Although related, P49 and P35 inhibit initiator and effector caspases, respectively, during infection of permissive insect cells. The molecular basis of this novel caspase specificity is unknown. To advance strategies for selective inhibition of the cell death caspases, we investigated biochemical differences between these baculovirus substrate inhibitors. We report here that P49 and P35 use similar mechanisms for stoichiometric inhibition that require caspase cleavage of their reactive site loops (RSL) and chemical contributions of a conserved N-terminal cysteine to stabilize the resulting inhibitory complex. Our data indicated that P49 functions as a homodimer that simultaneously binds two caspases. In contrast, P35 is a monomeric, monovalent inhibitor. P49 and P35 also differ in their RSL caspase recognition sequences. We tested the role of the P(4)-P(1) recognition motif for caspase specificity by monitoring virus-induced proteolytic processing of Sf-caspase-1, the principal effector caspase of the host insect Spodoptera frugiperda. When P49's TVTD recognition motif was replaced with P35's DQMD motif, P49 was impaired for inhibition of the initiator caspase that cleaves and activates pro-Sf-caspase-1 and instead formed a stable inhibitory complex with active Sf-caspase-1. In contrast, the effector caspase specificity of P35 was unaltered when P35's DQMD motif was replaced with TVTD. We concluded that the TVTD recognition motif is required but not sufficient for initiator caspase inhibition by P49. Our findings demonstrate a critical role for the P(4)-P(1) recognition site in caspase specificity by P49 and P35 and indicate that additional determinants are involved in target selection.
Institute for Molecular Virology and Department of Biochemistry, Graduate School and College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Michael P
MP
Guy
Paul D
PD
Friesen
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000502:physiology*; D001665:Binding Sites; D061945:Caspase Inhibitors; D053470:Caspases, Effector / Q000378:metabolism; D053471:Caspases, Initiator / Q000378:metabolism; D002460:Cell Line; D019281:Dimerization; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera; D013379:Substrate Specificity; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D061945:Caspase Inhibitors; D051034:Inhibitor of Apoptosis Proteins; D014764:Viral Proteins; C086430:inhibitor of apoptosis, Nucleopolyhedrovirus; D053470:Caspases, Effector; D053471:Caspases, Initiator