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The brace helices of MLKL mediate interdomain communication and oligomerisation to regulate cell death by necroptosis

Cell Death & Differentiationvolume 25pages15671580 (2018) | Download Citation


The programmed cell death pathway, necroptosis, relies on the pseudokinase, Mixed Lineage Kinase domain-Like (MLKL), for cellular execution downstream of death receptor or Toll-like receptor ligation. Receptor-interacting protein kinase-3 (RIPK3)-mediated phosphorylation of MLKL’s pseudokinase domain leads to MLKL switching from an inert to activated state, where exposure of the N-terminal four-helix bundle (4HB) ‘executioner’ domain leads to cell death. The precise molecular details of MLKL activation, including the stoichiometry of oligomer assemblies, mechanisms of membrane translocation and permeabilisation, remain a matter of debate. Here, we dissect the function of the two ‘brace’ helices that connect the 4HB to the pseudokinase domain of MLKL. In addition to establishing that the integrity of the second brace helix is crucial for the assembly of mouse MLKL homotrimers and cell death, we implicate the brace helices as a device to communicate pseudokinase domain phosphorylation event(s) to the N-terminal executioner 4HB domain. Using mouse:human MLKL chimeras, we defined the first brace helix and adjacent loop as key elements of the molecular switch mechanism that relay pseudokinase domain phosphorylation to the activation of the 4HB domain killing activity. In addition, our chimera data revealed the importance of the pseudokinase domain in conferring host specificity on MLKL killing function, where fusion of the mouse pseudokinase domain converted the human 4HB + brace from inactive to a constitutive killer of mouse fibroblasts. These findings illustrate that the brace helices play an active role in MLKL regulation, rather than simply acting as a tether between the 4HB and pseudokinase domains.

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Additional information

These authors contributed equally: Katherine A. Davies, Maria C. Tanzer.

Joint senior authors: John Silke, James M. Murphy.


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We thank staff at the Australian Synchrotron SAXS/WAXS beamline for assistance with data collection. We acknowledge support from an AINSE Postgraduate Research Award and an Australian Government Research Training Program Scholarship for KAD; a Victorian International Research Scholarship to MCT; RQ was supported by a scholarship from the Walter and Eliza Hall Institute as part of the International Student Program in Research Experience. We are grateful to the National Health and Medical Research Council for fellowship (PEC, 1079700; JS, 1058190; JMM, 1105754), grant (1057905; 1124735) and infrastructure (IRIISS 9000433) support; the Australian Research Council (Future Fellowship to MDWG, FT140100544); and to the Victorian Government Operational Infrastructure Support scheme.

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Author notes

  1. Edited by E. Baehrecke


  1. The Walter & Eliza Hall Institute of Medical Research, Parkville, VIC, Australia

    • Katherine A. Davies
    • , Maria C. Tanzer
    • , Samuel N. Young
    • , Rui Qin
    • , Emma J. Petrie
    • , Peter E. Czabotar
    • , John Silke
    •  & James M. Murphy
  2. Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia

    • Katherine A. Davies
    • , Maria C. Tanzer
    • , Emma J. Petrie
    • , Peter E. Czabotar
    • , John Silke
    •  & James M. Murphy
  3. Department of Biochemistry & Molecular Biology, The University of Melbourne, The Bio21 Institute, Parkville, VIC, Australia

    • Michael D. W. Griffin
    •  & Yee Foong Mok
  4. School of Pharmaceutical Sciences, Tsinghua University, Beijing, China

    • Rui Qin


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Conflict of interest

J.M.M., J.S., P.E.C., E.J.P. and S.N.Y. contribute to a project funded by Anaxis Pharma to develop necroptosis inhibitors. The remaining authors declare that they have no conflict of interest.

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Correspondence to John Silke or James M. Murphy.

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