Necroptosis is a form of programmed cell death distinguished from the better established cell death pathway, apoptosis, by lack of caspase activity and a loss of plasma membrane integrity. Morphologically similar to necrosis, in the act of necroptosis, the plasma membrane is disrupted, causing release of cellular components to the extracellular fluid and an ensuing inflammatory response. Necroptosis proceeds via a regulated kinase cascade involving Receptor Interacting Protein Kinases RIPK1 and RIPK3. Mixed Lineage Kinase domain-Like protein (MLKL), a pseudokinase, is the final known obligate effector essential for the execution of necroptosis. Whilst the MLKL pseudokinase domain is incapable of catalysing phosphotransfer reactions, it is the site of RIPK3 phosphorylation. This phosphorylation event is thought to be integral to flipping a molecular switch regulated by the pseudokinase domain, resulting in activation of MLKL. Upon activation, MLKL oligomerises and translocates to the plasma membrane, and is there thought to play a destabilising role. Details of MLKL’s molecular mechanism of action, such as the stoichiometry of oligomerisation and how it interacts with the plasma membrane, remain unknown.
We have investigated MLKL’s interactions with membranes using cryo-electron microscopy (CryoEM). An oligomeric mouse MLKL construct and small uni-lammellar vesicles with plasma membrane-like composition were used as a model system to study the MLKL:membrane interaction. The CryoEM results, coupled with data from liposome dye release assays, suggest that MLKL does not form large pores in the membrane. Direct visualisation of the MLKL:membrane interaction by CryoEM provides a useful lens with which to interpret models of MLKL membrane permeabilisation suggested by other biochemical techniques.