Structural basis of IAP recognition by Smac/DIABLO

Abstract

Apoptosis is an essential process in the development and homeostasis of all metazoans. The inhibitor-of-apoptosis (IAP) proteins suppress cell death by inhibiting the activity of caspases; this inhibition is performed by the zinc-binding BIR domains of the IAP proteins. The mitochondrial protein Smac/DIABLO promotes apoptosis by eliminating the inhibitory effect of IAPs through physical interactions. Amino-terminal sequences in Smac/DIABLO are required for this function, as mutation of the very first amino acid leads to loss of interaction with IAPs and concomitant loss of Smac/DIABLO function. Here we report the high-resolution crystal structure of Smac/DIABLO complexed with the third BIR domain (BIR3) of XIAP. Our results show that the N-terminal four residues (Ala-Val-Pro-Ile) in Smac/DIABLO recognize a surface groove on BIR3, with the first residue Ala binding a hydrophobic pocket and making five hydrogen bonds to neighbouring residues on BIR3. These observations provide a structural explanation for the roles of the Smac N terminus as well as the conserved N-terminal sequences in the Drosophila proteins Hid/Grim/Reaper. In conjunction with other observations, our results reveal how Smac may relieve IAP inhibition of caspase-9 activity. In addition to explaining a number of biological observations, our structural analysis identifies potential targets for drug screening.

Structural mechanisms of DIAP1 auto-inhibition and DIAP1-mediated inhibition of drICE

“…An IAP-binding motif, though not ostensibly abbreviated as IBM, was originally defined to contain four contiguous amino acids that resemble the Smac tetrapeptide AVPI [14][15][16] . This structurally defined motif, with binding affinities of 0.1-1 µM, has stringent requirement for the first (P1), third (P3) and fourth (P4) amino acids 13 .…”

“…7); this interaction locks caspase-9 in the inhibited state. During apoptosis, Smac/Diablo uses a similar tetrapeptide motif to occupy the BIR3 groove, hence releasing caspase-9 and relieving XIAP-mediated inhibition 14,15,27 . Caspase-3 or -7 is inhibited by an 18-residue peptide segment preceding the BIR2 domain of XIAP [8][9][10]28 .…”

“…Such negative regulation is countered by Smac/Diablo in mammals and RHG in fruit flies, which share a conserved tetrapeptide motif at their amino-termini 13 . This tetrapeptide binds a conserved surface groove on the BIR domains of IAPs and is known as the IAP-binding motif (IBM) [14][15][16] . Mutually exclusive binding to the BIR domains by IBMs present in either caspases or Smac/Diablo, and RHG is central for caspase regulation 5,13 .…”

XIAP: Cell death regulation meets copper homeostasis

“…Whether the activation of apoptosis is initiated by events that perturb the mitochondria (via caspase-9) or progress directly from cell surface receptors (via caspase-8), the ability of XIAP to inhibit the downstream executioner caspases-3 and -7 makes it a potent and broad inhibitor of cell death. Initiation of the apoptotic cascade involves the inactivation of XIAP through the release of the mitochondrial proteins Smac/DIABLO and Omi/HtrA2 into the cytoplasm where they bind to XIAP at precisely the same domains that mediate the interactions of XIAP with the caspases [17][18][19][20][21]. Acting as competitive inhibitors of caspase binding and through other mechanisms, these factors inhibit XIAP function, thereby facilitating propagation of the apoptotic cascade via the executioner caspases ( Figure 2).…”

IAPs – the ubiquitin connection

“…6 IAPs can be regulated by proapoptotic proteins, such as insect Reaper (Rpr), Grim, and HID, and mammalian Smac/ DIABLO and HtrA2/Omi, that bind to the IAPs' BIR domains via amino-terminal IAP binding motifs (IBMs). 7,8 XIAP was found to mediate ubiquitylation of Smac/DIABLO in vitro, 9 and DIAP1 was reported to cause ubiquitylation of the Drosophila IAP antagonists Grim, HID, and Rpr. 10 cIAP1 and cIAP2 were initially identified as proteins that bound to TRAF1 and TRAF2 in a complex associated with the cytoplasmic domain of TNF Receptor 2.…”