Differential affinity of FLIP and procaspase 8 for FADD’s DED binding surfaces regulates DISC assembly

Abstract

Death receptor activation triggers recruitment of FADD, which via its death effector domain (DED) engages DEDs in procaspase 8 and its inhibitor FLIP to form death-inducing signalling complexes (DISCs). The DEDs of FADD, FLIP and procaspase 8 interact with one another using two binding surfaces defined by α1/α4 and α2/α5 helices respectively. Here we report that FLIP has preferential affinity for the α1/α4 surface of FADD, whereas procaspase 8 has preferential affinity for FADD’s α2/α5 surface. These relative affinities contribute to FLIP being recruited to the DISC at comparable levels to procaspase 8 despite lower cellular expression. Additional studies, including assessment of DISC stoichiometry and functional assays, suggest that following death receptor recruitment, the FADD DED preferentially engages FLIP using its α1/α4 surface and procaspase 8 using its α2/α5 surface; these tripartite intermediates then interact via the α1/α4 surface of FLIP DED1 and the α2/α5 surface of procaspase 8 DED2.

Death effecter domain for the assembly of death-inducing signaling complex

“…An independent study on Fas DISC by Schleich et al [20] also showed that caspase-8/ cFLIP is more abundant than FADD, but the ratio was about five in western blot and two in mass spectrometry. In another study by Majkut et al [21], there found approximately two caspase-8/cFLIP molecules recruited for every FADD molecule in TRAIL DISC. Even though the ratio between FADD and caspase-8/cFLIP in DISC is significantly varied among these three independent studies, the common observation is that the adaptor protein FADD exists in a substantially smaller amount than the sum of tandem-DED-containing proteins, caspase-8 and cFLIP.…”

“…This feature contrasts CARD-CARD binding which is based on the electrostatic interaction between H2 and H3 face (not H2-H5 face) and H1-H4 face as observed in the crystal structure of CARD-CARD complex from Apaf-1 and procaspase-9 [8]. The crystal structure of MC159-vFLIP was used as a reference for the homology modeling studies on the tandem DEDs in procaspase-8 and cFLIP, which predicted very high structural similarity between them [19,21]. Especially, the involvement of H2-H5 face should be the characteristic element of DED-DED interaction, because the alternative H2-H3 face, instead, is commonly involved in CARD-CARD and in DD-DD interactions [8,16,27].…”

“…Recently, several biochemical studies have provided new insights into the interaction between DED-containing proteins in DISC [19][20][21]. In these studies, quantitative analyses of mass spectrometry and western blot were used to inspect the stoichiometry of DISC component proteins.…”

“…This view is well consistent with the previous studies in which DEDs of procaspase-8/-10 and cFLIP was shown to associate with themselves and each other and, moreover, to form a filamentous structure when overexpressed [33,34]. In order to inspect the surfaces of the inter-molecular DED-DED interactions and finally to build their structural model, homology models of tandem DEDs in procaspase-8 and cFLIP were generated based on MC159-vFLIP crystal structure and then the mutational evaluation was carried out for the possible interface residues [19,21]. The crystal structure of MC159-vFLIP previously showed that two hydrophobic faces, H2-H5 face and H1-H4 face, are responsible for the intra-molecular DED-DED interaction for tandem DEDs [17].…”

“…Instead, the crystal structures of tandem DEDs in two viral homologs of cFLIP were determined to reveal the structural details of intramolecular DED-DED interactions [17,18], which provide the analogical insight for the inter-molecular DED-DED interactions in DISC between Fadd, procaspase-8/-10 and cFLIP. In addition, several biochemical analyses on the stoichiometry of DISC components have been also reported very recently [19][20][21]. From the synthesis of all these recent studies, an advanced structural image on DISC assembly has been emerging.…”