Poster Presentation The 47th Lorne Conference on Protein Structure and Function 2022

Human MLKL is maintained by RIPK3 in an inactive conformation prior to disengagement and cell death by necroptosis (#138)

Yanxiang Meng 1 , Sarah E Garnish 1 , Katherine A Davies 1 , Akiko Koide 2 , Eric Denbaum 2 , Annette V Jacobsen 1 , Wayland Yeung 3 , Andre L Samson 1 , Christopher R Horne 1 , Cheree Fitzgibbon 1 , Samuel N Young 1 , Cindy Luo 1 , Lung-Yu Liang 1 , Angus D Cowan 1 , Pheobe P C Smith 1 , Andrew I Webb 1 , Emma J Petrie 1 , Joanne M Hildebrand 1 , Guillaume Lessene 1 , Jarrod J Sandow 1 , Natarajan Kannan 3 , Peter E Czabotar 1 , Shohei Koide 2 , James M Murphy 1
  1. Walter and Eliza Hall Institute for Medical Research, Parkville, VICTORIA, Australia
  2. Perlmutter Cancer Cener, New York University Langone Health, New York, NY, United States
  3. Institute of Bioinformatics, University of Georgia, Athens, GA, USA

Necroptosis is a caspase-independent form of programmed cell death that results in the compromise of plasma membranes and release of inflammatory cellular contents. Dysregulated necroptosis has been shown to play a role in a range of different human pathologies, including ischemia-reperfusion injury, inflammatory diseases, and inflammatory bowel disease. Phosphorylation of MLKL by the RIPK3 kinase leads to MLKL oligomerization, translocation to, and permeabilization of, the plasma membrane to induce necroptotic cell death. The precise choreography of MLKL activation remains incompletely understood. Here, we used Monobodies, synthetic binding proteins, that bind the pseudokinase domain of MLKL to detect endogenous protein interactions within human cells. We showed that MLKL is stably bound by RIPK3 prior to their disengagement upon necroptosis induction. Crystal structures of MLKL pseudokinase domain in complex with two different monobodies or RIPK3 kinase domain identified two distinct conformations of MLKL pseudokinase domain. These structures support that human RIPK3 maintains MLKL in an inactive conformation prior to the induction of necroptosis. These studies provide further evidence that MLKL undergoes a large conformational change upon activation and identify MLKL disengagement from RIPK3 as a key regulatory step in the necroptosis pathway.