The MLKL Channel in Necroptosis Is an Octamer Formed by Tetramers in a Dyadic Process

Abstract

Oligomerization of the mixed-lineage kinase domain-like protein (MLKL) is essential for its cation channel function in necroptosis. Here we show that the MLKL channel is an octamer comprising two previously identified tetramers most likely in their side-by-side position. Intermolecule disulfide bonds are present in the tetramer but are not required for octamer assembly and necroptosis. MLKL forms oligomers in the necrosome and is then released from the necrosome before or during its membrane translocation. We identified two MLKL mutants that could not oligomerize into octamers, although they formed a tetramer, and also, one MLKL mutant could spontaneously form a disulfide bond-linked octamer. Subsequent analysis revealed that the tetramers fail to translocate to the plasma membrane and that the MLKL octamer formation depends on ␣-helices 4 and 5. While MLKL could be detected from outside the cells, its N-and C-terminal ends could not be detected, indicating that the MLKL octamer spans across the plasma membrane, leaving its N and C termini inside the cell. These data allowed us to propose a 180°symmetry model of the MLKL octamer and conclude that the fully assembled MLKL octamers, but not the previously described tetramers, act as effectors of necroptosis.

MLKL forms disulfide bond-dependent amyloid-like polymers to induce necroptosis

“…2H, Right). MLKL formed complexes of more than 669 kDa, much larger than a tetramer (∼240 KDa) or the recently reported octamer (∼480 kDa) (26). Phosphorylated MLKL eluted solely in the large complexes (lanes 11 and 12), which contained MLKL polymers (Fig.…”

“…(xi) N-terminal-tagged MLKL cannot form polymers, providing a possible explanation for its inability to induce necroptosis, shown previously (19,26).…”

“…N-terminal-tagged MLKL fails to form large polymers and cannot induce necroptosis (19,26). These results suggest that the exact N-terminal sequences are important for polymer formation in the cells.…”

“…It was reported that N-terminal-tagged MLKL could not induce necroptosis (20,26), but the reason is not well understood. C-terminal Flag-tagged or N-terminal Flag-tagged MLKL was overexpressed in MLKL-knockout HeLa cells.…”

“…Others reported that MLKL binds to a calcium transporter or sodium transporters on the cell membrane to induce ion influx (19,20). Recently it was reported that recombinant MLKL can form cation channels (25,26). How to reconcile these results and clarify the execution step of necroptosis remains a big challenge.…”