Autoinhibitory control of MLKL governs pseudokinase domain phosphorylation and oligomerization during necroptosis

Necroptotic cell death triggers the release of inflammatory mediators but the exact mechanisms controlling its activation are not fully understood. Previous studies have identified key steps during necroptosis, which are believed to be coupled: MLKL phosphorylation by RIPK3, release of N-terminal autoinhibition, and MLKL oligomerization. Yet, ectopic expression of phosphomimetic MLKL is insufficient to induce necroptosis in human cells. Here, we employ five different pharmacological, biological, and genetic methods to demonstrate that inhibiting the MLKL N terminus prevents both phosphorylation and oligomerization. Conversely, loss of interaction between the N-terminal four-helical bundle and brace domains demonstrates basal MLKL phosphorylation, even in the absence of necroptotic stimuli. Moreover, we show that MLKL phosphorylation is not necessary for maintaining MLKL oligomer stability. We propose that MLKL is released from autoinhibition prior to phosphorylation, explaining why phosphomimetic MLKL lacks cytotoxic activity.