Necroptosis is a cell death mechanism characterised by permeabilisation of the inner plasma membrane, with subsequent release of cellular contents inviting an inflammatory response. The process is dependent on the kinase activity of Receptor Interacting Protein Kinase (RIPK)-1 and RIPK-3, which assemble into a poorly-characterised amyloid-like structure called the ‘necrosome’. The pseudokinase, Mixed Lineage Kinase-domain Like (MLKL), is recruited to the necrosome and phosphorylated by RIPK3 promoting oligomer formation, which is essential for necroptosis.
MLKL is a multi-domain protein in which the N-terminal four-helix bundle (4HB) executes cell death via lipid engagement and is tethered to the C-terminal pseudokinase domain (PsKD) by a two-helix linker. The PsKD is the molecular switch that constrains the 4HB, while the linker facilitates oligomerisation upon activation. The molecular details behind how RIPK3 engagement flips the molecular switch to activate MLKL, along with the arrangement and stoichiometry of the human MLKL (hMLKL) oligomer remain unclear.
To understand how the PsKD governs activation of hMLKL, we studied activity of recombinant hMLKL in vitro and in cells by reconstituting these mutations in MLKL-/- cell lines. We identified PsKD mutants with defective activity that remains monomeric, but transition from an inactive to active state. We characterised the monomer:tetramer transition using a suite of biophysical techniques, including AUC, SAXS and MD, in combination with Mass Spectrometry, to experimentally map rearrangements within the PsKD of the inert MLKL monomer to the activated tetramer. Assembly of oligomers upon membrane engagement was further characterised using Fast-Scan Atomic Force Microscopy. Together, this work has advanced our knowledge of the molecular switch mechanism of the MLKL PsKD in regulating cell death, and how it can be pharmacologically targeted in inflammatory pathologies.