The BIR domain of IAP-like protein 2 is conformationally unstable: implications for caspase inhibition

Abstract

Several IAP (inhibitor of apoptosis) proteins regulate cell fate decisions, and the X-linked IAP (XIAP) does so in part by inhibiting caspases, proteases that execute the apoptotic pathway. A tissue-specific homologue of XIAP, known as ILP2 (IAP-like protein 2), has previously been implicated in the control of apoptosis in the testis by direct inhibition of caspase 9. In examining this protein we found that the putative caspase 9 interaction domain is a surprisingly weak inhibitor and is also conformationally unstable. Comparison with the equivalent domain in XIAP demonstrated that the instability is due to the lack of a linker segment N-terminal to the inhibitory BIR (baculovirus IAP repeat) domain. Fusion of a 9-residue linker from XIAP to the N-terminus of ILP2 restored tight caspase 9 inhibition, dramatically increased conformational stability and allowed crystallization of the ILP2 BIR domain in a form strikingly similar to the XIAP third BIR domain. We conclude that ILP2 is an unstable protein, and cannot inhibit caspase 9 in a physiological way on its own. We speculate that ILP2 requires assistance from unidentified cellular factors to be an effective inhibitor of apoptosis in vivo.

IAP proteins: sticking it to Smac

“…In this issue of the Biochemical Journal two reports [11,12], as well as a recent study from our laboratory [13], support the overall e2 C. S. Duckett conclusion of the study by Silke et al [10], but approach the question through the analysis not of XIAP, but of three distinct protective IAPs. The study by Vucic et al [11] is an elegant structural characterization of ML-IAP (melanoma IAP [14]), and the study by Shin et al [12] examines the structural and functional properties of human ILP-2 (IAP-like protein 2; also known as BIRC8 [15,16]) and our group has examined the protective properties of a baculoviral IAP (Op-IAP [17]).…”

“…The study by Vucic et al [11] is an elegant structural characterization of ML-IAP (melanoma IAP [14]), and the study by Shin et al [12] examines the structural and functional properties of human ILP-2 (IAP-like protein 2; also known as BIRC8 [15,16]) and our group has examined the protective properties of a baculoviral IAP (Op-IAP [17]). The first study shows that, despite its strongly protective properties, ML-IAP is significantly inferior to XIAP in terms of caspase inhibition, but has a very high affinity for Smac.…”

Caspase inhibitors: viral, cellular and chemical

“…In this way, XIAP inhibits caspase 9 with an IC50 of around 10 nM. 27,28 Like XIAP, cIAP1 and cIAP2 have three BIRs and a RING domain. However, although cIAP1 and cIAP2 are able to bind to caspases, they are not able to inhibit them in physiological circumstances, because their K i are greater than 5 mM, 29 so their ability to inhibit apoptosis is probably indirect rather than by the direct inhibition of caspase activity.…”

Challenge and promise: roles for Livin in progression and therapy of cancer

“…The Biochemical Journal and The Journal of Biological Chemistry report further investigations that reveal the reason why low affinity exists between some IAPs and caspases (35,36). One is ILP-2, which, similar to Livin, is a weak caspase-9 inhibitor.…”