Reactive-Site Cleavage Residues Confer Target Specificity to Baculovirus P49, a Dimeric Member of the P35 Family of Caspase Inhibitors

The baculoviruses are large double-stranded DNA viruses of insects that trigger widespread apoptosis. To block host cell apoptosis and thereby enhance multiplication, these viruses encode diverse suppressors of apoptosis (reviewed in references 5, 6, and 14). P49 and P35 are two baculovirus-encoded apoptotic suppressors that function by inhibiting a broad range of the cell death proteases known as caspases (2, 3, 19, 30, 40, 45). Although related to one another, P49 and P35 display different caspase specificities in the infected insect cell (21, 22, 45). Because selective inhibition of caspases may be advantageous in therapeutics for apoptosis-associated diseases, the molecular basis of target specificity by P49 and P35 is of considerable interest. The caspases are a family of cysteinyl aspartate-specific proteases that are critical effectors of apoptosis in metazoans (reviewed in references 15, 23, 32, and 33). Caspase-mediated proteolysis promotes cellular disassembly that includes chromatin condensation, nuclear DNA cleavage, membrane blebbing, and cell fragmentation. Thus, these death proteases are subject to regulation by diverse cellular and viral mechanisms (4, 6, 15, 33, 34, 36). Upon apoptotic signaling, initiator caspases are autoactivated through interactions of their N-terminal prodomain with specific adaptor proteins (1, 31, 35, 42). Subsequently, the effector caspases are activated by initiator caspase-mediated cleavage of their inactive zymogen (procaspase) form. Procaspase cleavage occurs between the large and small subunit domains, allowing for the assembly of the active protease, consisting of a dimer of heterodimers with two active sites (15, 23, 36). The substrate specificity of initiator caspases differs from that of effector caspases, due in part to their unique functions during execution of apoptosis (37). Not surprisingly, because of their central role in cell death, both types of caspases are important therapeutic targets for the treatment of apoptosis-associated diseases (11, 23). The novel selectivity of P49 and P35 for different caspases in insects provides a unique opportunity to define the in vivo determinants of caspase specificity in the experimentally advantageous system provided by the baculovirus-infected cell. Studying baculovirus caspase inhibitors has led to important insights into the role and regulation of caspases in apoptosis (4, 5, 7, 14). In general, a single baculovirus species encodes only one functional inhibitor, either a substrate inhibitor or an inhibitor of apoptosis protein (IAP). P35 from the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) is the founding member of the family of substrate inhibitors that includes baculovirus P49 and entomopoxvirus P33 (3, 9, 27). Sequence comparisons have also revealed P35 homologs in Spodoptera littoralis NPV (SlNPV), Spodoptera litura NPV, Leucania separata NPV, and Bombyx mori NPV (reviewed in reference 5). The potency of P35 as a caspase inhibitor is attributed to its novel solvent-exposed reactive-site loop (RSL), which is readily recognized and cleaved at Asp87 by the target caspase (2, 3, 24, 30, 40, 43). Cleavage of the P4-P1 recognition motif DQMD87↓G (see Fig. ​Fig.1A),1A), located at the apex of the RSL, triggers a conformational change that positions the P35 N terminus in the caspase active site to prevent peptide hydrolysis and thereby form a stable complex (8, 10, 12, 24, 40). The inhibited complex consists of the caspase homodimer with each of the two active sites occupied by a separate monomer of P35. FIG. 1. Requirement of Cys2 for caspase inhibition by P49. (A) Comparison of P49 and P35. P49 (446 residues) and P35 (299 residues) share significant amino acid sequence identity. Cleavage of P49 and P35 occurs at Asp94 and Asp87, respectively, within the caspase ... P49 is a stoichiometric substrate inhibitor with P35-like properties. First discovered in SlNPV (9), P49 has the capacity to inhibit both effector and initiator caspases (19, 29, 45). Cleavage of P49 at an aspartate residue (Asp94) within the caspase recognition motif TVTD94↓G (see Fig. ​Fig.1A)1A) is necessary for formation of an inhibitory complex with the target caspase (19, 29, 45). Although the structure of P49 is unknown, sequence alignments suggest that it resembles that of P35, including the presence of a prominent RSL that presents Asp94 for cleavage (29, 45). Thus, we predicted that P49 functions by using a P35-like mechanism for caspase inhibition. P49 prevents proteolytic processing of effector caspases Sf-caspase-1 and Sf-caspase-2 during baculovirus infection of the moth Spodoptera frugiperda (order Lepidoptera) (45). Caspase cleavage of P49 at TVTD94↓G is required for P49-mediated suppression of virus-induced apoptosis. Thus, P49 is a substrate inhibitor of the initiator caspase designated Sf-caspase-X, which is responsible for the proteolysis and activation of Sf-caspase-1 and -2 (45). In a cellular context, Sf-caspase-X is also inhibited by baculovirus Op-IAP, but not P35, which fails to inactivate other initiator caspases, including those from invertebrates (16, 21, 28, 39, 45). Because P49's TVTD recognition motif resembles the caspase processing sites TETD↓G and AETD↓G of pro-Sf-caspase-1 and -2, respectively, we hypothesized that P49's in vivo specificity is determined by its caspase recognition motif. To test this possibility, we altered the recognition motifs of P49 and P35, delivered the modified caspase inhibitors to Spodoptera cells by using recombinant baculoviruses, and monitored proteolytic processing of Sf-caspase-1 and -2 during infection. We report here that, when P49's TVTD motif was swapped with P35's DQMD motif, P49 was impaired as an initiator caspase inhibitor and instead functioned downstream as an effector caspase inhibitor. In contrast, when P35's DQMD motif was swapped with TVTD, P35's selectivity for effector caspases was unaltered. Thus, P49 requires a TVTD motif for Spodoptera initiator caspase inhibition, but this motif alone is insufficient to confer the same activity upon P35. To search for additional determinants of caspase selectivity, we compared the biochemical properties of P49 and P35. By using recombinant P49, produced and purified from Escherichia coli for the first time, we determined that P49 and P35 use comparable mechanisms for caspase inhibition. In contrast to P35, which acts as a monomer, P49 functions as a homodimer with the capacity to form a stable complex with two individual caspase dimers. Thus, P49 is the first example of a divalent caspase inhibitor in which caspase targeting can be altered by sequence alterations in the caspase recognition motif.