A Highly Conserved Arginine Is Critical for the Functional Folding of Inhibitor of Apoptosis (IAP) BIR Domains

Abstract

The inhibitor of apoptosis (IAP) proteins are found in all animals and regulate apoptosis (programmed cell death) by binding and inhibiting caspase proteases. This inhibition is overcome by several apoptosis stimulators, including Drosophila Hid and mammalian Smac/DIABLO, which bind to 65-residue baculovirus IAP repeat (BIR) domains found in one to three copies in all IAPs. Virtually all BIRs contain three Cys and a His that bind zinc, a Gly in a tight turn, and an Arg. The functional and structural role of the Arg was investigated in isolated BIR domains from the baculovirus Orgyia pseudotsugata Op-IAP and the Drosophila DIAP1 proteins. Mutation of the Arg to either Ala or Lys abolished Hid and Smac binding to BIRs, despite the Hid/Smac binding site being located on the opposite side of the BIR domain from the Arg. The mutant BIR domains also exhibited weakened zinc binding, increased sensitivity to limited proteolysis, and altered circular dichroism spectra indicative of perturbed domain folding. Examination of known BIR structures indicates that the Arg side chain makes simultaneous bridging hydrogen bonds and a cation-pi interaction for which the Arg guanidino group is uniquely well suited. These interactions are likely critical for stabilizing the tertiary fold of BIR domains in all IAPs, explaining the conservation of this residue.

Insight into autoproteolytic activation from the structure of cephalosporin acylase: A protein with two proteolytic chemistries

“…4). Similar interactions are recognized as crucial for the structural stabilization of caspase BIR domain (28) and neutrophil collagenase (29). Moreover, (Phe-177) is the only residue of 682 modeled amino acid residues in a disallowed region of the Ramachandran plot in all mutant crystal structures as well as in precursor and mature CA (see Fig.…”

A dual role for the API2 moiety in API2-MALT1-dependent NF-κB activation: heterotypic oligomerization and TRAF2 recruitment

“…This region encompasses the C-terminal two-thirds of the BIR1 domain, which contains the conserved CX 2 CX 16 HX 6À8 C motif capable of coordinating a zinc ion. This conserved 'zinc-finger' structure underlies the surface groove where caspases bind to IAP BIR domains (Luque et al, 2002). It is tempting to speculate that this zinc finger may mediate oligomerization by interacting in some way with the caspase-like domain of the MALT1 moiety.…”

Survivin interacts with Smac/DIABLO in ovarian carcinoma cells but is redundant in Smac-mediated apoptosis

“…Smac is capable of binding to the BIR domains of XIAP, c-IAP1, c-IAP2, Melanoma IAP (ML-IAP), and OpIAP from the baculovirus O. pseudotsugata [10,21,47,48], and an interaction with survivin was originally suggested but could not be proven unambiguously [11]. The single BIR domain of survivin bears a close structural homology to BIR2 of XIAP [49], including the critical arginine residue required for functional folding [50]. Recently, it was suggested that Smac and survivin can bind to each other, an interaction essential for inhibition of Taxolmediated apoptosis in HeLa cells [51].…”