Oral Presentation 23rd Annual Lorne Proteomics Symposium 2018

The MLKL pseudokinase domain controls protein activity and oligomer formation during necroptotic cell death (#70)

Jarrod J Sandow 1 , Emma Petrie 1 , Annette Jacobsen 1 , Michael Griffin 2 , Brian Smith 3 , Isabelle Lucet 1 , Katherine Davies 1 , John Silke 1 , Peter Czabotar 1 , Richard Henderson 4 , James Murphy 1 , Andrew Webb 1
  1. Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia
  2. Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, Australia
  3. School of Molecular Sciences, La Trobe University, Bundoora, VIC, Australia
  4. Department of Pharmacology, Cambridge University, Cambridge, UK

Necroptosis is a cell death mechanism characterised by permeabilisation of the inner plasma membrane, with subsequent release of cellular contents initiating an inflammatory response. The pseudokinase, Mixed Lineage Kinase-domain Like (MLKL) is the most terminal known effector of necroptotic cell death. 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. Following cell death stimuli, MLKL is phosphorylated by Receptor Interacting Protein Kinase-3 promoting activation and oligomer formation, which is essential for necroptosis. The stoichiometry, structure and dynamics of MLKL during transition to an active oligomer remains unclear. Using native mass spectrometry, we determined that MLKL assembles as a tetramer where as a mutant MLKL (E351KMLKL) remains monomeric. Assembly of tetramers upon membrane engagement was further characterised using Fast-Scan Atomic Force Microscopy. To determine the structural changes between monomeric E351KMLKL and tetrameric MLKL, we utilised cross-linking mass spectrometry with constraint mapping and small angle x-ray scattering to model the monomer and tetramer MLKL structures. By combining these results with hydrogen-deuterium exchange mass spectrometry we mapped the dynamic changes that occur as MLKL transitions to an active state. This analysis revealed that the PsKD is the molecular switch that constrains the 4HB, while the linker facilitates oligomerisation upon activation. We next validated our observations by reconstituting MLKL-/- cell lines with mutant MLKL proteins designed to disrupt interaction sites identified from our analysis. Together, this work describes how the PsKD of MLKL regulates the transition to an active tetramer during necroptotic cell death and identifies potential pharmacological targets in inflammatory pathologies.