Oral Presentation The 44th Lorne Conference on Protein Structure and Function 2019

The conformational state of the MLKL protein tightly regulates necroptotic cell death (#36)

Emma J Petrie 1 , Jarrod J Sandow 1 , Annette V Jacobsen 1 , Lung-Yu Liang 1 , Wilhamus JA Kersten 1 , Michael D Griffin 2 , Brian J Smith 3 , Diane Coursier 1 , Cheree Fitzgibbon 1 , Katherine Davies 1 , Samuel N Young 1 , Ahmad Wardak 1 , Guilaume Lessene 1 , John Silke 1 , Peter Czabotar 1 , Andrew I Webb 1 , Gerrad Manning 4 , Isabelle S Lucet 1 , James M Murphy 1
  1. The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia
  2. Biochemistry and Molecular Biology, The Bio21 Institute, The University of Melbourne, Parkville, Vic, Australia
  3. Biochemistry and Molecular Biology, La Trobe University, Bundoora, Victoria, Australia
  4. Department of Bioinformatics and Computational Biology, Genentech Inc, South San Francisco, California, United States

The Mixed-linage kinase-domain Like (MLKL) protein is essential to the programmed cell death mechanism of necroptosis.  During necroptosis the inner plasma membrane is compromised, leading to cell swelling and release of intracellular contents, inviting an inflammatory response.

MLKL is the most terminal protein in the kinase signalling cascade that leads to necroptosis, which is well characterised downstream of the TNF receptor.  Phosphorylation of the C-terminal pseudokinase domain (PsKD) of MLKL by RIPK3 is thought to release the N-terminal 4-helical bundle (4HB), which is the killing domain, and promote oligomer formation.

The detection of phosphorylated MLKL oligomers at the plasma membrane is a hallmark of necroptosis.  While recombinant MLKL can disrupt liposomes in vitro, little is known about the mechanism of action including how MLKL transitions from an inert cytoplasmic protein to a killer assembly and the arrangement of MLKL subunits within the oligomer.

This talk will describe how the PsKD controls the conformational state of MLKL.  Through coupling mass-spectrometry and biophysical techniques we characterised the conformation of the basal cytosolic monomer and the structural changes that occur as MLKL transitions to a tetrameric assembly that kills cells.  We characterised PsKD mutants associated with some cancers that stabilise the basal state of MLKL.  When reconstituted in MLKL-/- cells these mutations impacted the kinetics of tetramer formation and delayed cell death suggesting a delay in necroptosis may have a growth advantage for some tumours.

This work also highlighted species-specific differences in RIPK3-mediated activation of MLKL. We have identified viral MLKL-like proteins that target this interaction to block necroptosis, which may in part explain the tangential evolution of MLKL through different phyla.