Biochemical and structural characterization of the baculovirus inhibitor of apoptosis repeat domains

The mammalian gene family encoding inhibitors of apoptosis (IAPs) are intrinsic, cellular suppressors of programmed cell death. Their primary, but not exclusive, role is in direct interaction with and inhibition of caspases, cysteine proteases that are specifically activated in the apoptotic cascade. Regulation of IAP expression and function, therefore, is fundamental to the control of apoptosis. X-linked IAP (XIAP), the best-characterized IAP, is a potent inhibitor of caspases-3/7/9. The inhibitory activity of XIAP is mapped to two different domains, the BIR2 and BIR3 domains. BIR2 inhibits caspase-3/7 while BIR3 inhibits caspase-9. Although the structural basis for BIR2 selectivity and inhibition has been recently elucidated, it remains to be determined how BIR3 inhibits caspase-9. This dissertation focused on characterizing the interaction between XIAP-BIR3 domain and its targets, caspase-9 and Smac.; By dissecting XIAP-BIR3 domain, we identified three components that are critical for caspase-9 inhibition: (1) residues in the Smac groove, (2) His-343 of the fifth α-helix and (3) the N-terminus of the BIR3 domain. We showed that BIR2 could be converted into a caspase-9 inhibitor by introducing the first two components. Importance of the third component is demonstrated by revealing that BIR3 lacking the N-terminus linker and ILP2 are not caspase-9 inhibitor. The crystal structure of X-ILP2 BIR/Smac served as a tool to analyze the structural basis of recognition between BIR3 and caspase-9. Finally, we provide evidence for the molecular and structural basis for Smac-mediated IAP neutralization.