The mixed lineage kinase domain-like protein (MLKL) is responsible for plasma membrane (PM) permeabilization during the last stage of necroptosis, a pro-inflammatory programmed cell death pathway. MLKL contains three domains: the pseudokinase domain (PsKD), a brace region, and a four-helical bundle (4HB). Phosphorylation of PsKD residues S345 and S347 is essential for MLKL oligomerization as it triggers conformational changes that expose the 4HB, which enables protein oligomerization and PM permeabilization. Understanding the molecular mechanisms of this allosteric signal is critical for the rational design of ligands to modulate necroptosis in the context of neurodegenerative and inflammatory diseases. Here, we performed all-atom molecular dynamics simulations of the wild-type, phosphorylated, and mutant MLKL proteoforms to investigate the conformational changes underlying 4HB exposure. Markov state modeling revealed three dominant macrostates corresponding to the open, transition, and closed conformations. Hydrogen-bond network analysis with a novel clustering approach uncovered a switch in allosteric pathways in the open state, driven by enhanced motion of two PsKD helices. Based on this model, we identified and computationally tested an MLKL mutant that facilitates 4HB exposure and could potentially favor protein oligomerization, thereby compromising plasma membrane integrity.
Ramirez et al. (Sun,) studied this question.