title: Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death.
abstract: Necroptosis is considered to be complementary to the classical caspase-dependent programmed cell death pathway, apoptosis. The pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) is an essential effector protein in the necroptotic cell death pathway downstream of the protein kinase Receptor Interacting Protein Kinase-3 (RIPK3). How MLKL causes cell death is unclear, however RIPK3-mediated phosphorylation of the activation loop in MLKL trips a molecular switch to induce necroptotic cell death. Here, we show that the MLKL pseudokinase domain acts as a latch to restrain the N-terminal four-helix bundle (4HB) domain and that unleashing this domain results in formation of a high-molecular-weight, membrane-localized complex and cell death. Using alanine-scanning mutagenesis, we identified two clusters of residues on opposing faces of the 4HB domain that were required for the 4HB domain to kill cells. The integrity of one cluster was essential for membrane localization, whereas MLKL mutations in the other cluster did not prevent membrane translocation but prevented killing; this demonstrates that membrane localization is necessary, but insufficient, to induce cell death. Finally, we identified a small molecule that binds the nucleotide binding site within the MLKL pseudokinase domain and retards MLKL translocation to membranes, thereby preventing necroptosis. This inhibitor provides a novel tool to investigate necroptosis and demonstrates the feasibility of using small molecules to target the nucleotide binding site of pseudokinases to modulate signal transduction.
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Departments of Medical Biology and.
authors.forename: Joanne M
authors.initials: JM
authors.lastname: Hildebrand
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Departments of Medical Biology and.
authors.forename: Maria C
authors.initials: MC
authors.lastname: Tanzer
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Departments of Medical Biology and Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, VIC 3800, Australia;
authors.forename: Isabelle S
authors.initials: IS
authors.lastname: Lucet
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia;
authors.forename: Samuel N
authors.initials: SN
authors.lastname: Young
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia;
authors.forename: Sukhdeep K
authors.initials: SK
authors.lastname: Spall
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Departments of Medical Biology and.
authors.forename: Pooja
authors.initials: P
authors.lastname: Sharma
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia;
authors.forename: Catia
authors.initials: C
authors.lastname: Pierotti
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Departments of Medical Biology and.
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Garnier
authors.affiliation: Biomolecular Interactions Centre, School of Biological Sciences, University of Canterbury, Christchurch, New Zealand; Department of Biochemistry, Bio21 Institute, University of Melbourne, Parkville, VIC 3052, Australia;
authors.forename: Renwick C J
authors.initials: RC
authors.lastname: Dobson
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Departments of Medical Biology and.
authors.forename: Andrew I
authors.initials: AI
authors.lastname: Webb
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Departments of Medical Biology and.
authors.forename: Anne
authors.initials: A
authors.lastname: Tripaydonis
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Departments of Medical Biology and.
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Babon
authors.affiliation: Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia; and.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Mulcair
authors.affiliation: Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia; and.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Scanlon
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Departments of Medical Biology and.
authors.forename: Warren S
authors.initials: WS
authors.lastname: Alexander
authors.affiliation: SynThesis MedChem, Parkville, VIC 3052, Australia.
authors.forename: Andrew F
authors.initials: AF
authors.lastname: Wilks
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Departments of Medical Biology and.
authors.forename: Peter E
authors.initials: PE
authors.lastname: Czabotar
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Departments of Medical Biology and Pharmacology and Therapeutics, University of Melbourne, Parkville, VIC 3050, Australia;
authors.forename: Guillaume
authors.initials: G
authors.lastname: Lessene
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Departments of Medical Biology and jamesm@wehi.edu.au silke@wehi.edu.au.
authors.forename: James M
authors.initials: JM
authors.lastname: Murphy
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Departments of Medical Biology and jamesm@wehi.edu.au silke@wehi.edu.au.
authors.forename: John
authors.initials: J
authors.lastname: Silke
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D020128:Inhibitory Concentration 50; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009154:Mutation; D009336:Necrosis; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; C570694:MLKL protein, mouse; D011494:Protein Kinases; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C485615:Ripk3 protein, mouse
keywords: ATP mimetic; RIP kinase; programmed necrosis; pseudoenzyme