Mechanistic Insights into MLKL Activation via Allosteric Pathways Identified Through Molecular Models

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), the brace region, and the four-helical bundle (4HB). Phosphorylation of PsKD at amino acids S345 and S347 is essential for MLKL function as it triggers conformational changes that expose the 4HB and enables protein oligomerization and sub-sequent PM permeabilization. Understanding the molecular mechanisms of this allosteric signal is critical for the rational design of ligands that modulate cell death via necroptosis as terapeutic alternatives for neurodegenerative and inflammatory disorder. We simulated the wild-type, phosphorylated, and mutant MLKL proteoforms to build a Markov state model that revealed three dominant macrostates corresponding to the open, transition, and closed conformations. Hydrogen-bond/Hydrophobic network analysis, with a novel clustering approach, identified a switch in the allosteric path-ways that favors the open state. Based on this model, we identified and tested computationally an MLKL mutant that facilitates 4HB exposure, and could favor protein oligomerization and compromise plasma membrane integrity. We present a comprehensive all-atom molecular dynamics study to characterize the conformational changes that enable exposure of the 4HB at the molecular level.