Eukaryotic cells can undergo different forms of programmed cell death, many of which culminate in plasma membrane rupture as the defining terminal event1,2,3,4,5,6,7. Plasma membrane rupture was long thought to be driven by osmotic pressure, but it has recently been shown to be in many cases an active process, mediated by the protein ninjurin-18 (NINJ1). Here we resolve the structure of NINJ1 and the mechanism by which it ruptures membranes. Super-resolution microscopy reveals that NINJ1 clusters into structurally diverse assemblies in the membranes of dying cells, in particular large, filamentous assemblies with branched morphology. A cryo-electron microscopy structure of NINJ1 filaments shows a tightly packed fence-like array of transmembrane α-helices. Filament directionality and stability is defined by two amphipathic α-helices that interlink adjacent filament subunits. The NINJ1 filament features a hydrophilic side and a hydrophobic side, and molecular dynamics simulations show that it can stably cap membrane edges. The function of the resulting supramolecular arrangement was validated by site-directed mutagenesis. Our data thus suggest that, during lytic cell death, the extracellular α-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. The membrane protein NINJ1 is therefore an interactive component of the eukaryotic cell membrane that functions as an in-built breaking point in response to activation of cell death.
This work was supported by the Deutsche Forschungsgemeinschaft under Germany’s Excellence Strategy EXC 2075–390740016 and the Stuttgart Center for Simulation Science (SC SimTech) to K.P., by ERC-CoG 770988 (InflamCellDeath) and SNF Project funding (310030B_198005, 310030B_192523) to P.B., by the Swiss Nanoscience Institute and the Swiss National Science Foundation via the NCCR AntiResist (180541) to S.H. and the NCCR Molecular Systems Engineering (51NF40-205608) to D.J.M., by the Helmholtz Young Investigator Program of the Helmholtz Association to C.S., by the SNF Professorship funding (PP00P3_198903) to C.P., EMBO postdoctoral fellowship ALTF 27-2022 to E.H. and by the Scientific Service Units of IST Austria through resources provided by the NMR and Life Science Facilities to P.S. Molecular dynamics simulations were performed on the HoreKa supercomputer funded by the Ministry of Science, Research and the Arts Baden-Württemberg and by the Federal Ministry of Education and Research. The authors thank the BioEM Lab of the Biozentrum, University of Basel for support; V. Mack, K. Shkarina and J. Fricke for technical support; D. Ricklin and S. Vogt for peptide synthesis; P. Pelczar for support with animals; S.-J. Marrink and P. Telles de Souza for supply with Martini3 parameters and scripts; and P. Radler und M. Loose for help with QCM. Fig. 4g and Extended Data Fig. 1a were in part created with BioRender.com.
Degen M, Santos JC, Pluhackova K, et al. Structural basis of NINJ1-mediated plasma membrane rupture in cell death. Nature. 2023. doi:10.1038/s41586-023-05991-z
Degen, M., Santos, J. C., Pluhackova, K., Cebrero, G., Ramos, S., Jankevicius, G., … Hiller, S. (2023). Structural basis of NINJ1-mediated plasma membrane rupture in cell death. Nature. Springer Nature. https://doi.org/10.1038/s41586-023-05991-z
Degen, Morris, José Carlos Santos, Kristyna Pluhackova, Gonzalo Cebrero, Saray Ramos, Gytis Jankevicius, Ella Hartenian, et al. “Structural Basis of NINJ1-Mediated Plasma Membrane Rupture in Cell Death.” Nature. Springer Nature, 2023. https://doi.org/10.1038/s41586-023-05991-z.
M. Degen et al., “Structural basis of NINJ1-mediated plasma membrane rupture in cell death,” Nature. Springer Nature, 2023.
Degen M, Santos JC, Pluhackova K, Cebrero G, Ramos S, Jankevicius G, Hartenian E, Guillerm U, Mari SA, Kohl B, Müller DJ, Schanda P, Maier T, Perez C, Sieben C, Broz P, Hiller S. 2023. Structural basis of NINJ1-mediated plasma membrane rupture in cell death. Nature.
Degen, Morris, et al. “Structural Basis of NINJ1-Mediated Plasma Membrane Rupture in Cell Death.” Nature, Springer Nature, 2023, doi:10.1038/s41586-023-05991-z.
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