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

Death receptor activation triggers recruitment of FADD, which via its death effector domain (DED) engages the DEDs of 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 alpha 1/alpha 4 and alpha 2/alpha 5 helices, respectively. Here we report that FLIP has preferential affinity for the alpha 1/alpha 4 surface of FADD, whereas procaspase 8 has preferential affinity for FADD's alpha 2/alpha 5 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 alpha 1/alpha 4 surface and procaspase 8 using its alpha 2/alpha 5 surface; these tripartite intermediates then interact via the alpha 1/alpha 4 surface of FLIP DED1 and the alpha 2/alpha 5 surface of procaspase 8 DED2.