Necroptosis is a lytic form of programmed cell death that is implicated in various inflammatory diseases. Signalling proceeds via a regulated kinase cascade involving Receptor Interacting Protein Kinases RIPK1 and RIPK3. RIPK3 phosphorylates the pseudokinase domain of Mixed Lineage Kinase-domain Like protein, MLKL, flipping a molecular switch which results in MLKL oligomerisation, translocation to, and permeabilisation of the plasma membrane, causing cell death. MLKL’s remarkable transformation from inert monomer to oligomeric, membrane associated killer is enabled by specialised domains: the N-terminal membrane permeabilising 4HB domain, the brace helices which are essential for oligomerisation, and the regulatory C-terminal pseudokinase domain.
Our previous work established that recombinant human MLKL forms a tetrameric oligomer1, whilst the mouse orthologue forms trimers2. Intriguingly, we recently discovered that the rat MLKL orthologue, which shares 86% sequence identity with mouse MLKL, forms tetramers like the human counterpart, as does the more distantly related pig MLKL. This suggested that mouse MLKL may be an outlier amongst mammalian MLKLs, at least in respect to its oligomer. In our current study, we have exploited the high degree of sequence conservation between rat and mouse MLKL to pinpoint which residues are responsible for determining MLKL oligomer stoichiometry. Our results highlight that the brace region and pseudokinase domain of MLKL influence oligomer stoichiometry, and that MLKL is surprisingly functionally adaptable.