Necroptosis is an inflammatory form of programmed cell death that is implicated in various human pathologies. 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 permeabilization 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.
We recently structurally characterized the conformational change that MLKL’s pseudokinase domain undergoes in response to phosphorylation. However, in the only published structure of full-length MLKL (the mouse orthologue), there are no contacts between MLKL’s pseudokinase and 4HB executioner domain, leading us to question how this conformational change could be propagated throughout the protein. In our current study, we report a new, more complete crystal structure of full‑length rat MLKL, with an extra helix in the 4HB resolved. This helix directly contacts the pseudokinase domain, and MLKL’s brace helices, revealing for the first time a direct nexus point between MLKL’s 3 domains, and establishing a possible mechanism for signal propagation. In addition, our novel structure verifies that the unusual arrangement of MLKL’s domains observed in the first structure is conserved across orthologues, and is independent of crystal packing, an open question in the field since the first structure was published in 2013.