Amphipathic N-terminal helices drive MLKL-mediated necroptosis through an antimicrobial peptide-like mechanism across...

Necroptosis is a highly regulated lytic cell death executed by the pseudokinase, MLKL. Although the N-terminal 4HB domain is essential for MLKL-mediated membrane disruption, it is poorly conserved within the animal kingdom. How MLKL targets and disrupts the plasma membrane, and whether these mechanisms are evolutionarily conserved, remain incompletely understood. Using human MLKL and a teleost homolog, we found that MLKL could be activated independent of the brace region, an interdomain linker facilitating MLKL oligomerization. Instead, the N-terminal 4HB alone was sufficient to drive membrane rupture, provided that its extreme N-terminus remained intact. Stepwise removal of the N-terminal residues abolished cell death despite enhanced oligomer formation, indicating that oligomerization was a separate event and insufficient for membrane lysis. We further demonstrated that electrostatic charges and hydrophobic residues coordinated the conformational changes to enable membrane targeting, oligomerization, and permeabilization, whereas disruption of either property markedly reduced cytotoxicity. Mechanistically, the N-terminal helices displayed physicochemical features resembling antimicrobial peptides and exhibited direct membrane lysis activity in both human and fish MLKL. Despite divergence in charge distribution and amphipathic properties among species, MLKL-mediated membrane lysis was evolutionarily conserved. Consistent with this, functional residues in the N-terminal pore-forming and the C-terminal pseudokinase domains were preserved between human and teleost MLKL, with mutations in the former causing loss of function, and mutations in the latter relieving autoinhibition and triggering constitutive activation. These findings collectively reveal the molecular determinants that govern MLKL-mediated cell death, highlighting how its machinery has structurally adapted while preserving its core function across evolution.