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Molecular assembly of the aerolysin pore reveals a swirling membrane-insertion mechanism

Abstract

Aerolysin is the founding member of a superfamily of β-pore–forming toxins whose pore structure is unknown. We have combined X-ray crystallography, cryo-EM, molecular dynamics and computational modeling to determine the structures of aerolysin mutants in their monomeric and heptameric forms, trapped at various stages of the pore formation process. A dynamic modeling approach based on swarm intelligence was applied, whereby the intrinsic flexibility of aerolysin extracted from new X-ray structures was used to fully exploit the cryo-EM spatial restraints. Using this integrated strategy, we obtained a radically new arrangement of the prepore conformation and a near-atomistic structure of the aerolysin pore, which is fully consistent with all of the biochemical data available so far. Upon transition from the prepore to pore, the aerolysin heptamer shows a unique concerted swirling movement, accompanied by a vertical collapse of the complex, ultimately leading to the insertion of a transmembrane β-barrel.

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Figure 1: Aerolysin structure and flexibility.
Figure 2: The prepore state trapped by the Y221G mutation.
Figure 3: Pre-stem loop is locked in the Y221G aerolysin mutant.
Figure 4: Multiple stages toward pore formation revealed by the K246C E258C mutant.
Figure 5: Near-atomistic model structure of the wild-type aerolysin pore.
Figure 6: Swirling mechanism promoting transition between the prepore and the pore conformation.

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Acknowledgements

We thank F. David and S. Ho for technical assistance, G. Knot and D. Demurtas for help with EM, M. Grütter for testing of crystals, C. Schulze-Briese for assistance in collecting the data and M. Schiltz for support with X-ray crystallography. EM computation was performed at the Vital-IT (Swiss Institute of Bioinformatics). This work was supported by the Swiss National Science Foundation with grants to M.D.P. (200021_122120 and 200020_138013) and to F.G.v.d.G. and by the Swiss Initiative for Systems Biology, SystemsX.ch to F.G.v.d.G. and H.S.

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Authors

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F.G.v.d.G. and M.D.P. designed and supervised the study. M.T.D. performed simulations and modeling. I.I. performed cryo-EM analysis, molecular biology, nanodiscs and biochemical experiments. L.P. performed protein production and X-ray crystallography. M.K., M.C. and H.S. supported the EM. M.T.D., I.I., F.G.v.d.G. and M.D.P. wrote the manuscript.

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Correspondence to F Gisou van der Goot or Matteo Dal Peraro.

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The authors declare no competing financial interests.

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Supplementary Text and Figures

Supplementary Results, Supplementary Table 1 and Supplementary Figures 1–9. (PDF 1725 kb)

Aerolysin pore formation via a swirling mechanism

The movie shows a top and a side view of a morphing from the prepore (Fig. 2) to the pore models (Fig. 5). Conversion from one protein arrangement to the other is possible via a swirling mechanism, which can take place without any relevant topological bottlenecks (Fig. 6). Aerolysin domain 1 is not shown in the movie for sake of clarity. (AVI 36819 kb)

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Degiacomi, M., Iacovache, I., Pernot, L. et al. Molecular assembly of the aerolysin pore reveals a swirling membrane-insertion mechanism. Nat Chem Biol 9, 623–629 (2013). https://doi.org/10.1038/nchembio.1312

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