Abstract
Necroptosis is a lytic programmed cell death pathway with origins in innate immunity that is frequently dysregulated in inflammatory diseases. The terminal effector of the pathway, MLKL, is licensed to kill following phosphorylation of its pseudokinase domain by the upstream regulator, RIPK3 kinase. Phosphorylation provokes the unleashing of MLKL’s N-terminal four-helix bundle (4HB or HeLo) domain, which binds and permeabilizes the plasma membrane to cause cell death. The precise mechanism by which the 4HB domain permeabilizes membranes, and how the mechanism differs between species, remains unclear. Here, we identify the membrane binding epitope of mouse MLKL using NMR spectroscopy. Using liposome permeabilization and cell death assays, we validate K69 in the α3 helix, W108 in the α4 helix, and R137/Q138 in the first brace helix as crucial residues for necroptotic signaling. This epitope differs from the phospholipid binding site reported for human MLKL, which comprises basic residues primarily located in the α1 and α2 helices. In further contrast to human and plant MLKL orthologs, in which the α3-α4 loop forms a helix, this loop is unstructured in mouse MLKL in solution. Together, these findings illustrate the versatility of the 4HB domain fold, whose lytic function can be mediated by distinct epitopes in different orthologs.
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Data availability
The NMR chemical shifts have been deposited in the BioMagResBank (BMRB) under the BMRB ID is 51061.
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Acknowledgements
We are grateful to the NMR facility (University of Melbourne) and the National Deuteration Facility for providing crucial infrastructure for this study. The University of Melbourne NMR facility was enabled by an Australian Research Council equipment grant LE120100022. We are grateful to the National Health and Medical Research Council for fellowship (JMH, 1142669; PEC, 1079700; JMM, 1172929), grant (1124735, 2002965) and infrastructure (IRIISS 9000719) support; Anaxis Pharma Pty Ltd for funding support; and the Victorian Government Operational Infrastructure Support scheme. We acknowledge Australian Government Research Training Program Stipend Scholarship support (to SEG and AVJ) and the Wendy Dowsett Scholarship (to SEG). The National Deuteration Facility is partly funded by the National Collaborative Research Infrastructure Strategy (NCRIS), an Australian Government initiative.
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AS and CRH designed and performed experiments, analyzed data and co-wrote the paper with JMM; KW carried out the biodeuteration of the recombinant protein for NMR; CF, KAD, SEG, AVJ, ALS, JMH, and AW performed experiments and analyzed data; PEC, EJP, PRG, and JMM supervised the project and contributed to experimental design and data analysis. All authors commented on the paper.
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CF, KAD, SEG, ALS, JMH, PEC, EJP, and JMM contribute to, or have contributed to, a project with Anaxis Pharma to develop necroptosis inhibitors. The remaining authors declare no competing interests.
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Mouse dermal fibroblasts were prepared using procedures approved by and conducted in accordance with the Animal Ethics Committee of the Walter and Eliza Hall Institute, Australia.
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Sethi, A., Horne, C.R., Fitzgibbon, C. et al. Membrane permeabilization is mediated by distinct epitopes in mouse and human orthologs of the necroptosis effector, MLKL. Cell Death Differ 29, 1804–1815 (2022). https://doi.org/10.1038/s41418-022-00965-6
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DOI: https://doi.org/10.1038/s41418-022-00965-6
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