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

Two novel structures of human RIPK3, in complex with necroptosis effector protein MLKL and kinase inhibitors (#231)

Katherine A Davies 1 2 , Yanxiang Meng 1 2 , Cheree Fitzgibbon 1 2 , Samuel N Young 1 2 , Sarah E Garnish 1 2 , Christopher R Horne 1 2 , Cindy Luo 1 2 , Jean-Marc Garnier 3 , Lung-Yu Liang 1 2 , Angus D Cowan 1 2 , Andre L Samson 1 2 , Guillaume Lessene 1 2 , Jarrod J Sandow 1 2 , Peter E Czabotar 1 2 , James M Murphy 1 2
  1. WEHI, Parkville, VIC, Australia
  2. Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia
  3. Anaxis Pharma Pty Ltd, Parkville, Victoria, Australia

Necroptosis is a form of programmed cell death characterised by lack of caspase activity and a loss of plasma membrane integrity. Morphologically similar to necrosis, during necroptosis, the plasma membrane is disrupted, causing release of cellular components to the extracellular fluid and an ensuing inflammatory response. Necroptosis proceeds via a regulated kinase cascade involving Receptor Interacting Protein Kinases RIPK1 and RIPK3. Upon phosphorylation by RIPK3, the pseudokinase MLKL becomes activated and oligomerises and translocates to the plasma membrane, which it disrupts, causing cell death.

Recent studies from our group suggest that under the steady state human RIPK3 and MLKL are pre-associated in a basal complex, which dissociates once MLKL is phosphorylated1, 2, 3. To further characterise this complex, we recently solved the first crystal structure of the human MLKL pseudokinase domain and human RIPK3 kinase domain complex, as well as the first crystal structure of the human RIPK3 kinase domain alone4. Human RIPK3 is a highly unstable protein and diffracting crystals were only obtained in the presence of Type I and Type II kinase inhibitors. From the two structures, we were able to examine the biology of the basal MLKL:RIPK3 complex and compare RIPK3 conformation alone and in complex with MLKL. Serendipitously, the inclusion of inhibitors in the structures revealed an unexpected binding mode for a Type II kinase inhibitor in the MLKL:RIPK3 complex, with interesting implications for therapeutics targeting RIPK3 and the necroptosis pathway.

  1. Garnish SE, et al. Conformational interconversion of MLKL and disengagement from RIPK3 precede cell death by necroptosis. Nature communications 12, 2211 (2021).
  2. Petrie EJ, et al. Conformational switching of the pseudokinase domain promotes human MLKL tetramerization and cell death by necroptosis. Nature communications 9, 2422 (2018).
  3. Davies KA, et al. Distinct pseudokinase domain conformations underlie divergent activation mechanisms among vertebrate MLKL orthologues. Nature communications 11, 3060-3060 (2020).
  4. Meng Y, et al. Human RIPK3 maintains MLKL in an inactive conformation prior to cell death by necroptosis. Nature communications, (2021; in press).