MLKL activity requires a splicing-regulated, druggable intramolecular interaction.

Necroptosis is an inflammatory form of regulated cell death implicated in a range of human pathologies, whose execution depends on the poorly understood pseudokinase mixed lineage kinase domain-like (MLKL). Here, we report that splicing-dependent insertion of a short amino acid sequence in the C-terminal α-helix (Hc) of MLKL abolishes cell killing activity and creates an anti-necroptotic isoform that counteracts cell death induced by the necroptosis-proficient protein in mice and humans. We show that interaction of Hc with a previously unrecognized hydrophobic groove is essential for necroptosis, which we exploited in a strategy to identify small molecules that inhibit MLKL and substantially ameliorate disease in murine models of necroptosis-driven dermatitis and abdominal aortic aneurysm. Thus, alternative splicing of microexons controls the ability of MLKL to undergo an intramolecular rearrangement essential for necroptosis with potential to guide the development of allosteric MLKL inhibitors for the treatment of human disease.