Poster Presentation The 46th Lorne Conference on Protein Structure and Function 2021

Mapping the signaling events underlying RIPK3 activation of MLKL during necroptosis (#307)

Sarah E Garnish 1 2 , Yanxiang Meng 1 2 , Akiko Koide 3 4 , Jarrod J Sandow 1 2 , Eric Denbaum 3 , Annette V Jacobsen 1 2 , Wayland Yeung 5 , Andre L Samson 1 2 , Christopher R Horne 1 2 , Cheree Fitzgibbon 1 , Samuel N Young 1 , Phoebe P.C Smith 1 , Andrew I Webb 1 2 , Emma J Petrie 1 2 , Joanne M Hildebrand 1 2 , Natarajan Kannan 5 , Peter E Czabotar 1 2 , Shohei Koide 3 6 , James M Murphy 1 2
  1. The Walter and Eliza Hall Institute, Parkville, VIC, Australia
  2. University of Melbourne, Department of Medical Biology, Parkville, Australia
  3. Perlmutter Cancer Center, New York University Langone Health, New York, NY 10016
  4. Department of Medicine, New York University School of Medicine, New York, NY 10016
  5. Institute of Bioinformatics, University of Georgia, Athens, GA 30602, USA
  6. Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016

Necroptosis is a lytic form of programmed cell death that is associated with multiple human pathologies including ischemia reperfusion injury, multiple sclerosis, and inflammatory bowel disease. Pseudokinase mixed-lineage kinase domain-like protein (MLKL) is the terminal effector of the necroptotic pathway. The current understanding is that receptor interacting serine/threonine protein kinase-3 (RIPK3)-mediated phosphorylation of MLKL triggers an oligomerisation event, promoting the translocation to, and permeabilization of the plasma membrane. However, the nature of MLKL interaction with RIPK3 and the precise choreography of MLKL activation remains incompletely understood. Our study uses synthetic binding proteins, termed Monobodies,  that bind the pseudokinase domain of MLKL in human cells to investigate the signalling events underlying RIPK3 activation of MLKL. Here, we present two pseudokinase domain targeting Monobodies, Monobody-32 and Monobody-27, that recognise distinct human MLKL pseudokinase domain conformers. Using these Monobodies, we show pre-association of endogenous human MLKL and RIPK3 and following necroptotic stimulation, MLKL disengagement from RIPK3. Crystal structures of these Monobodies in complex with human MLKL pseudokinase domain and mutational analyses of these binding epitopes elucidates a role for the MLKL activation loop in RIPK3 engagement. These studies provide further evidence that upon activation MLKL undergoes a conformational change which accompanies MLKL disengagement from RIPK3.