Article | Published:

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

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

Abstract

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Additional information

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

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

References

  1. 1.

    Murphy JM, Czabotar PE, Hildebrand JM, Lucet IS, Zhang JG, Alvarez-Diaz S, et al. The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. Immunity. 2013;39:443–53.

  2. 2.

    Sun L, Wang H, Wang Z, He S, Chen S, Liao D, et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell. 2012;148:213–27.

  3. 3.

    Wu J, Huang Z, Ren J, Zhang Z, He P, Li Y, et al. Mlkl knockout mice demonstrate the indispensable role of Mlkl in necroptosis. Cell Res. 2013;23:994–1006.

  4. 4.

    Zhao J, Jitkaew S, Cai Z, Choksi S, Li Q, Luo J, et al. Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis. Proc Natl Acad Sci USA. 2012;109:5322–7.

  5. 5.

    Silke J, Rickard JA, Gerlic M. The diverse role of RIP kinases in necroptosis and inflammation. Nat Immunol. 2015;16:689–97.

  6. 6.

    Rickard JA, O’Donnell JA, Evans JM, Lalaoui N, Poh AR, Rogers TW, et al. RIPK1 regulates RIPK3-MLKL driven systemic inflammation and emergency hematopoiesis. Cell. 2014;157:1175–88.

  7. 7.

    Rickard JA, Anderton H, Etemadi N, Nachbur U, Darding M, Peltzer N, et al. TNFR1-dependent cell death drives inflammation in Sharpin-deficient mice. Elife 2014;3:e03464.

  8. 8.

    Dannappel M, Vlantis K, Kumari S, Polykratis A, Kim C, Wachsmuth L, et al. RIPK1 maintains epithelial homeostasis by inhibiting apoptosis and necroptosis. Nature. 2014;513:90–4.

  9. 9.

    Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, et al. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol. 2005;1:112–9.

  10. 10.

    Linkermann A, Brasen JH, Darding M, Jin MK, Sanz AB, Heller JO, et al. Two independent pathways of regulated necrosis mediate ischemia-reperfusion injury. Proc Natl Acad Sci USA. 2013;110:12024–9.

  11. 11.

    Muller T, Dewitz C, Schmitz J, Schroder AS, Brasen JH, Stockwell BR, et al. Necroptosis and ferroptosis are alternative cell death pathways that operate in acute kidney failure. Cell Mol Life Sci. 2017;74:3631–45.

  12. 12.

    Luedde M, Lutz M, Carter N, Sosna J, Jacoby C, Vucur M, et al. RIP3, a kinase promoting necroptotic cell death, mediates adverse remodelling after myocardial infarction. Cardiovasc Res. 2014;103:206–16.

  13. 13.

    Gautheron J, Vucur M, Reisinger F, Cardenas DV, Roderburg C, Koppe C, et al. A positive feedback loop between RIP3 and JNK controls non-alcoholic steatohepatitis. EMBO Mol Med. 2014;6:1062–74.

  14. 14.

    Gunther C, He GW, Kremer AE, Murphy JM, Petrie EJ, Amann K, et al. The pseudokinase MLKL mediates programmed hepatocellular necrosis independently of RIPK3 during hepatitis. J Clin Invest. 2016;126:4346–60.

  15. 15.

    Wang H, Sun L, Su L, Rizo J, Liu L, Wang LF, et al. Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3. Mol Cell. 2014;54:133–46.

  16. 16.

    Li J, McQuade T, Siemer AB, Napetschnig J, Moriwaki K, Hsiao YS, et al. The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis. Cell. 2012;150:339–50.

  17. 17.

    Newton K, Wickliffe KE, Maltzman A, Dugger DL, Strasser A, Pham VC, et al. RIPK1 inhibits ZBP1-driven necroptosis during development. Nature. 2016;540:129–33.

  18. 18.

    Rodriguez DA, Weinlich R, Brown S, Guy C, Fitzgerald P, Dillon CP, et al. Characterization of RIPK3-mediated phosphorylation of the activation loop of MLKL during necroptosis. Cell Death Differ. 2016;23:76–88.

  19. 19.

    Tanzer MC, Tripaydonis A, Webb AI, Young SN, Varghese LN, Hall C, et al. Necroptosis signalling is tuned by phosphorylation of MLKL residues outside the pseudokinase domain activation loop. Biochem J. 2015;471:255–65.

  20. 20.

    Cook WD, Moujalled DM, Ralph TJ, Lock P, Young SN, Murphy JM, et al. RIPK1- and RIPK3-induced cell death mode is determined by target availability. Cell Death Differ. 2014;21:1600–12.

  21. 21.

    Cai Z, Jitkaew S, Zhao J, Chiang HC, Choksi S, Liu J, et al. Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. Nat Cell Biol. 2014;16:55–65.

  22. 22.

    Chen X, Li W, Ren J, Huang D, He WT, Song Y, et al. Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death. Cell Res. 2014;24:105–21.

  23. 23.

    Hildebrand JM, Tanzer MC, Lucet IS, Young SN, Spall SK, Sharma P, et al. Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death. Proc Natl Acad Sci USA. 2014;111:15072–7.

  24. 24.

    Quarato G, Guy CS, Grace CR, Llambi F, Nourse A, Rodriguez DA, et al. Sequential engagement of distinct MLKL phosphatidylinositol-binding sites executes necroptosis. Mol Cell. 2016;61:589–601.

  25. 25.

    Dondelinger Y, Declercq W, Montessuit S, Roelandt R, Goncalves A, Bruggeman I, et al. MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates. Cell Rep. 2014;7:971–81.

  26. 26.

    Tanzer MC, Matti I, Hildebrand JM, Young SN, Wardak A, Tripaydonis A, et al. Evolutionary divergence of the necroptosis effector MLKL. Cell Death Differ. 2016;23:1185–97.

  27. 27.

    Huang D, Zheng X, Wang ZA, Chen X, He WT, Zhang Y, et al. The MLKL channel in necroptosis is an octamer formed by tetramers in a dyadic process. Mol Cell Biol. 2017;37:5.

  28. 28.

    Petrie EJ, Hildebrand JM, Murphy JM. Insane in the membrane: a structural perspective of MLKL function in necroptosis. Immunol Cell Biol. 2017;95:152–9.

  29. 29.

    Murphy JM, Lucet IS, Hildebrand JM, Tanzer MC, Young SN, Sharma P, et al. Insights into the evolution of divergent nucleotide-binding mechanisms among pseudokinases revealed by crystal structures of human and mouse MLKL. Biochem J. 2014;457:369–77.

  30. 30.

    Su L, Quade B, Wang H, Sun L, Wang X, Rizo J. A plug release mechanism for membrane permeation by MLKL. Structure. 2014;22:1489–1500.

  31. 31.

    Jacobsen AV, Lowes KN, Tanzer MC, Lucet IS, Hildebrand JM, Petrie EJ, et al. HSP90 activity is required for MLKL oligomerisation and membrane translocation and the induction of necroptotic cell death. Cell Death Dis. 2016;7:e2051.

  32. 32.

    Brumatti G, Ma C, Lalaoui N, Nguyen NY, Navarro M, Tanzer MC, et al. The caspase-8 inhibitor emricasan combines with the SMAC mimetic birinapant to induce necroptosis and treat acute myeloid leukemia. Sci Transl Med. 2016;8:339ra369.

  33. 33.

    Murphy JM, Vince JE. Post-translational control of RIPK3 and MLKL mediated necroptotic cell death. F1000Res. 2015;4:F1000.

  34. 34.

    Chen W, Zhou Z, Li L, Zhong CQ, Zheng X, Wu X, et al. Diverse sequence determinants control human and mouse receptor interacting protein 3 (RIP3) and mixed lineage kinase domain-like (MLKL) interaction in necroptotic signaling. J Biol Chem. 2013;288:16247–61.

  35. 35.

    Bigenzahn JW, Fauster A, Rebsamen M, Kandasamy RK, Scorzoni S, Vladimer GI, et al. An inducible retroviral expression system for tandem affinity purification mass-spectrometry-based proteomics identifies mixed lineage kinase domain-like protein (MLKL) as an heat shock protein 90 (HSP90) client. Mol Cell Proteom. 2016;15:1139–50.

  36. 36.

    Zhao XM, Chen Z, Zhao JB, Zhang PP, Pu YF, Jiang SH, et al. Hsp90 modulates the stability of MLKL and is required for TNF-induced necroptosis. Cell Death Dis. 2016;7:e2089.

  37. 37.

    Murphy JM, Metcalf D, Young IG, Hilton DJ. A convenient method for preparation of an engineered mouse interleukin-3 analog with high solubility and wild-type bioactivity. Growth Factors. 2010;28:104–10.

  38. 38.

    Hercus TR, Barry EF, Dottore M, McClure BJ, Webb AI, Lopez AF, et al. High yield production of a soluble human interleukin-3 variant from E. coli with wild-type bioactivity and improved radiolabeling properties. PLoS ONE. 2013;8:e74376.

  39. 39.

    Schuck P. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys J. 2000;78:1606–19.

  40. 40.

    Laue TM, Shah, BD, Ridgeway, TM & Pelletier, SL Analytical ultracentrifugation in biochemistry and polymer science. In: SE Harding AJRJCH (ed). Royal Society of Chemistry, 1992.

  41. 41.

    Kirby NM, Mudie ST, Hawley AM, Cookson DJ, Mertens HDT, Cowieson N, et al. A low-background-intensity focusing small-angle X-ray scattering undulator beamline. J Appl Crystallogr. 2013;46:1670–80.

  42. 42.

    Konarev PV, Volkov VV, Sokolova AV, Koch MHJ, Svergun DI. PRIMUS: A Windows PC-based system for small-angle scattering data analysis. J Appl Crystallogr. 2003;36:1277–82.

  43. 43.

    Svergun DI. Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J Appl Crystallogr. 1992;25:495–503.

  44. 44.

    Petoukhov MV, Svergun DI. Global rigid body modeling of macromolecular complexes against small-angle scattering data. Biophys J. 2005;89:1237–50.

Download references

Acknowledgements

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.

Author information

Author notes

  1. Edited by E. Baehrecke

Affiliations

  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

Authors

  1. Search for Katherine A. Davies in:

  2. Search for Maria C. Tanzer in:

  3. Search for Michael D. W. Griffin in:

  4. Search for Yee Foong Mok in:

  5. Search for Samuel N. Young in:

  6. Search for Rui Qin in:

  7. Search for Emma J. Petrie in:

  8. Search for Peter E. Czabotar in:

  9. Search for John Silke in:

  10. Search for James M. Murphy in:

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.

Corresponding authors

Correspondence to John Silke or James M. Murphy.

Electronic supplementary material

About this article

Publication history

Received

Revised

Accepted

Published

DOI

https://doi.org/10.1038/s41418-018-0061-3

Further reading