Residue Y362 is crucial for FLIPL to impart catalytic activity to pro-caspase-8 to suppress necroptosis.

The pro-form of caspase-8 prevents necroptosis by functioning in a proteolytically active complex with its catalytic-dead homolog, FLICE (FADD [Fas-associated death domain]-like interleukin 1β-converting enzyme)-like inhibitory protein long-form (FLIP_L). However, how FLIP_L imparts caspase-8 the catalytic activity to suppress necroptosis remains elusive. Here, we show that the protease-like domain of FLIP_L is essential for the activity of the caspase-8-FLIP_L heterodimer in blocking necroptosis. While substitution of two amino acids whose difference may contribute to the pseudo-caspase property of FLIP_L with the corresponding amino acids in caspase-8 does not restore the protease activity of FLIP_L, one of the amino acid replacements, tyrosine (Y) 362 to cysteine (C), is sufficient to completely abolish the activity of the caspase-8-FLIP_L heterodimer in cleaving receptor-interacting protein 1 (RIP1), thus releasing the necroptosis blockade. Unconstrained necroptosis is observed in embryonic day (E)10.5-E11.5 embryos of FLIP_L-Y362C knockin mice. Collectively, these results reveal that the protease-like domain of FLIP_L has a special structure that imparts the pro-caspase-8-FLIP_L heterodimer a unique catalytic activity toward RIP1 to prevent necroptosis.