A conserved XIAP-interaction motif in caspase-9 and Smac/DIABLO regulates caspase activity and apoptosis

X-linked inhibitor-of-apoptosis protein (XIAP) interacts with caspase-9 and inhibits its activity(1-3), whereas Smac (also known as DIABLO) relieves t his inhibition through interaction with XIAP(4-7). Here we show that XIAP a ssociates with the active caspase-9-Apaf-1 holoenzyme complex through bindi ng to the amino terminus of the linker peptide on the small subunit of casp ase-9, which becomes exposed after proteolytic processing of procaspase-9 a t Asp 315. Supporting this observation, point mutations that abrogate the p roteolytic processing but not the catalytic activity of caspase-9, or delet ion of the linker peptide, prevented caspase-9 association with XIAP and it s concomitant inhibition. We note that the N-terminal four residues of casp ase-9 linker peptide share significant homology with the N-terminal tetra-p eptide in mature Smac and in the Drosophila proteins Hid/Grim/Reaper(8,9), defining a conserved class of IAP-binding motifs. Consistent with this find ing, binding of the caspase-9 linker peptide and Smac to the BIR3 domain of XIAP is mutually exclusive, suggesting that Smac potentiates caspase-9 act ivity by disrupting the interaction of the linker peptide of caspase-9 with BIR3. Our studies reveal a mechanism in which binding to the BIR3 domain b y two conserved peptides, one from Smac and the other one from caspase-9, h as opposing effects on caspase activity and apoptosis.