Direct Activation of Human MLKL by a Select Repertoire of Inositol Phosphate Metabolites

Necroptosis is an inflammatory form of programmed cell death executed through plasma membrane rupture by the pseudokinase mixed lineage kinase domain-like (MLKL). We previously showed that MLKL activation requires metabolites of the inositol phosphate (IP) pathway. Here we reveal that I(1,3,4,6)P , I(1,3,4,5,6)P , and IP promote membrane permeabilization by MLKL through directly binding the N-terminal executioner domain (NED) and dissociating its auto-inhibitory region. We show that IP and inositol pentakisphosphate 2-kinase (IPPK) are required for necroptosis as IPPK deletion ablated IP production and inhibited necroptosis. The NED auto-inhibitory region is more extensive than originally described and single amino acid substitutions along this region induce spontaneous necroptosis by MLKL. Activating IPs bind three sites with affinity of 100-600 μM to destabilize contacts between the auto-inhibitory region and NED, thereby promoting MLKL activation. We therefore uncover MLKL's activating switch in NED triggered by a select repertoire of IP metabolites.