A SINGLE BIR DOMAIN OF XIAP SUFFICIENT FOR INHIBITING CASPASES

The inhibitor of apoptosis proteins (IAPs) constitute an evolutionaril y conserved family of homologous proteins that suppress apoptosis indu ced by multiple stimuli. Some IAP family proteins, including XIAP, cIA P-1, and cIAP-2, can bind and directly inhibit selected caspases, a gr oup of intracellular cell death proteases. These caspase-inhibiting IA P family proteins all contain three tandem BIR domains followed by a R ING zinc finger domain. To determine the structural basis for caspase inhibition by XIAP, we analyzed the effects of various fragments of th is IAP family protein on caspase activity in vitro and on apoptosis su ppression in intact cells. The RING domain of XIAP failed to inhibit t he activity of recombinant caspases-3 or -7, whereas a fragment of XIA P encompassing the three tandem BIR domains potently inhibited these c aspases in vitro and blocked Fas (CD95)-induced apoptosis when express ed in cells. Further dissection of the XIAP protein demonstrated that only the second of the three BIR domains (BIR2) was capable of binding and inhibiting these caspases. The apparent inhibition constants (K-i ) for BIR2-mediated inhibition of caspases-3 and -7 were 2-5 nM, indic ating that this single BIR domain possesses potent anti caspase activi ty, Expression of the BIR2 do main in cells also partially suppressed Fas-induced apoptosis and blocked cytochrome c-induced processing of c aspase-9 in cytosolic extracts, whereas BIR1 and BIR3 did not. These f indings identify BIR2 as the minimal caspase-inhibitory domain of XIAP and indicate that a single BIR domain can be sufficient for binding a nd inhibiting caspases.