Neuronal apoptosis-inhibitory protein does not interact with Smac and requires ATP to bind caspase-9

The (n) under bar euronal (a) under bar poptosis- (i) under bar nhibitory (p) under bar rotein (NAIP) is the founding member of the mammalian family of (i) under bar nhibitor of (ap) under bar optosis (IAP) proteins (also known as BIRC proteins) and has been shown to be antiapoptotic both in vivo and in vitro. The 160-kDa NAIP contains three distinct regions: an amino-terminal cluster of three (b) under bar aculoviral (i) under bar nhibitory (r) under bar epeat (BIR) domains, a central (n) under bar ucleotide binding (o) under bar ligomerization (d) under bar omain (NOD), and a carboxyl-terminal (l) under bar eucine-(r) under bar ich (r) under bar epeat (LRR) domain. The presence of the NOD and LRR domains renders NAIP unique among the IAPs and suggests that NAIP activity is regulated in a manner distinct from that of other members of the family. In this report, we examined the interaction of various regions of NAIP with caspase-9 and Smac. Recombinant NAIPs with truncations of the carboxyl-terminal LRR or NOD-LRR regions bound to caspase-9. In contrast, the full-length protein did not, suggesting some form of structural autoregulation. However, the association of the wild type full-length protein with caspase-9 was observed when interaction analysis was performed in the presence of ATP. Furthermore, mutation of the NAIP ATP binding pocket allowed full-length protein to interact with caspase-9. Thus, we conclude that NAIP binds to caspase-9 with a structural requirement for ATP and that in the absence of ATP the LRR domain negatively regulates the caspase-9-inhibiting activity of the BIR domains. Interestingly, and in contrast to the X-chromosome-linked inhibitor of apoptosis protein (XIAP), NAIP-mediated inhibition of caspase-9 was not countered by a peptide containing an amino-terminal (I) under bar AP (b) under bar inding (m) under bar otif (IBM). Consistent with this observation was the failure of Smac protein to interact with the NAIP BIR domains. These results demonstrate that NAIP is distinct from the other IAPs, both in demonstrating a ligand-dependent caspase-9 interaction and in demonstrating a distinct mechanism of inhibition.