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

Journal name:
Nature Chemical Biology
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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.

At a glance


  1. Aerolysin structure and flexibility.
    Figure 1: Aerolysin structure and flexibility.

    (a) Structure of monomeric pro-aerolysin (PDB code 1PRE). Structural domains are color coded as follows: domain 1 in gray, domain 2 in orange, domain 3 in green and domain 4 in yellow. The CTP is shown in blue, and the pre-stem domain, enlarged in the inset, is shown in tan. Residues involved in binding to the receptor are highlighted on domain 1 (binding to N-glycans on the receptors) and 2 (binding to the glycan core of GPI-anchored receptors). Positions of relevant mutations are highlighted in sticks: Tyr221 mutated to glycine produces a prepore state; cross-linking of pre-stem residues Lys246 and Glu258 (inset) produces a quasi-pore conformation. (b) Comparison of the flexibility of wild-type (WT) aerolysin and the Y221G mutant. The first eigenvector extracted by a principal component analysis of molecular dynamics simulations is projected on Cα atoms of the molecular dynamics–averaged structures. Their relative length and direction quantify the entity of domain flexibility when the CTP is removed from its position at domain 4 (Supplementary Fig. 1b).

  2. The prepore state trapped by the Y221G mutation.
    Figure 2: The prepore state trapped by the Y221G mutation.

    (a) Examples of side (top) and top (bottom) EM views of the Y221G mutant particles, as seen by negative staining (left), and their respective class averages (right). Scale bars, 10 nm. (b) Top and side views of the docking of seven Y221G monomers into the EM map at 16.6-Å resolution (Supplementary Fig. 2a,c). The positions of key interface residues and residues involved in receptor binding are shown in yellow and green space-filling representation, respectively. In the inset, the pre-stem loop (red) is adjacent to the low electron density (apparent hole) observed in the EM map. (c) Modeling of aerolysin prepore onto the GPI-anchored protein CD52. Domain 1 binds N-linked sugars (in blue) located on CD52, whereas domain 2 binds a mannose on the GPI-anchor (red) conserved core. The X-ray structure of pro-aerolysin complexed with a mannose-6-phosphate molecule is used to generate the complex (Supplementary Fig. 3a and Supplementary Table 1). The location of both binding sites forces the current orientation the prepore heptamer at the membrane (in yellow) (Supplementary Fig. 3b). (d) In vitro kinetics of heptamerization of wild-type aerolysin (Aero) and selected monomer-monomer interface mutants indicated in b. Aerolysin (wild type and mutants) was activated at 4 °C to prevent oligomerization with insoluble trypsin. After removal of trypsin, the sample was shifted at 25 °C, and heptamerization was monitored by SDS-PAGE overnight (O/N) for ~12 h. The complete data for all the interface residues are reported in Supplementary Figure 4.

  3. Pre-stem loop is locked in the Y221G aerolysin mutant.
    Figure 3: Pre-stem loop is locked in the Y221G aerolysin mutant.

    (a) X-ray structures of monomeric pro-aerolysin in the wild-type and Y221G mutant conformation are superimposed (Supplementary Fig. 1a). Inset shows the different conformation adopted by residue Leu277 in either the wild-type (blue) or the Y221G mutant (gray) X-ray structures. The two β-strands connecting domains 3 and 4 to the pre-stem loop are shown with strand Leu219–Lys229 (in red) and Asn269–Val281 (in blue). Two spheres highlight the position of Leu277 (blue) and Tyr221 (red). (b) Evolution of the secondary structure during molecular dynamics simulations of the wild-type (left) and Y221G (right) protein in the absence of the CTP. Red and blue regions represent conservation of β-strand structure indicated in a. Upon removal of the CTP, unfolding propagates throughout the two highlighted β-strands toward the pre-stem domain in the wild type but stops at Gly221–Leu277 level in the Y221G mutant.

  4. Multiple stages toward pore formation revealed by the K246C E258C mutant.
    Figure 4: Multiple stages toward pore formation revealed by the K246C E258C mutant.

    (a) Side (top) and top (bottom) EM views of negative-stained heptameric K246C E258C mutant samples and class averages thereof. A stem structure, highlighted by a red arrow, is visible between the two asymmetric heptamers. Scale bars, 10 nm. (b) Cryo-EM map (18.3-Å resolution; Supplementary Fig. 2b,c) of the asymmetric K246C E258C oligomeric particles corresponding to two heptamers with different configurations (post-prepore in dark blue and quasi-pore in light blue) derived from cryo-EM particles (Supplementary Fig. 2b,c). (c) Comparison of cryo-EM maps of Y221G mutant heptamer (prepore in gray; Fig. 2b), and the post-prepore and quasi-pore conformations extracted from the K246C E258C mutant heptamer. A wide cavity is observed in the prepore lumen, which gradually fills up as the protein proceeds to the post-prepore and then quasi-pore. Transition of the post-prepore to the quasi-pore involves a collapse of the structure. (d) Negative stain of a wild-type heptameric aerolysin sample after centrifugation. Nonaggregated asymmetric double heptamers are circled in red and magnified in the insets. Scale bar, 100 nm.

  5. Near-atomistic model structure of the wild-type aerolysin pore.
    Figure 5: Near-atomistic model structure of the wild-type aerolysin pore.

    (a) Model of the aerolysin β-barrel, taking into account the boundaries established by cysteine-scanning analysis and planar lipid bilayer studies (Supplementary Fig. 8a). An aromatic belt (Tyr233, Trp265) is ideally positioned to anchor the structure at the lipid head group–acyl chain boundary. The positions of charged residues pointing into the barrel lumen are also indicated (Supplementary Fig. 8b). (b) Side and top views of the model of the membrane-inserted pore of aerolysin showing the arrangement of the seven monomers in the disk-like structure of the cryo-EM map of the K246C E258C quasi-pore. Positions are shown for the residues at the monomer-monomer interface that we experimentally showed to be involved in oligomerization (Fig. 2b,d and Supplementary Fig. 4). The position of the lipid bilayer, consistent with conformation of wild-type aerolysin in nanodiscs (Supplementary Fig. 7a), preliminary cryo-electron tomography (data not shown) and molecular dynamics simulation of the barrel (Supplementary Fig. 8b), is represented by the dashed lines.

  6. Swirling mechanism promoting transition between the prepore and the pore conformation.
    Figure 6: Swirling mechanism promoting transition between the prepore and the pore conformation.

    (a) Ribbon representations of aerolysin monomer as observed in the model of the prepore (transparent) and the membrane-inserted (solid) state. For clarity, domain 1 is not shown. During the prepore-to-pore transition state, domains 3 (green) and 4 (yellow) rotate and flatten to a position almost parallel to the membrane plane. During this transition, the pre-stem loop is extracted and flips around to form the transmembrane β-barrel (Supplementary Video 1). (b) Ribbon and space-filling (for two representative monomers) representation of the prepore and the membrane-inserted state, illustrating the overall swirling movement undergone by the complex, the change in the relative position of domains 4 in the heptamer and the increase in monomer-monomer contact surface (Supplementary Video 1). The blue space-filled monomer has the same orientation as in a.


  1. Aerolysin pore formation via a swirling mechanism
    Video 1: 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.

Accession codes

Primary accessions

Protein Data Bank

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Protein Data Bank



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Author information

  1. These authors contributed equally to this work.

    • Matteo T Degiacomi &
    • Ioan Iacovache


  1. Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

    • Matteo T Degiacomi &
    • Matteo Dal Peraro
  2. Global Health Institute, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

    • Ioan Iacovache,
    • Lucile Pernot &
    • F Gisou van der Goot
  3. Center for Cellular Imaging and NanoAnalytics, Biozentrum, University Basel, Basel, Switzerland.

    • Mohamed Chami,
    • Misha Kudryashev &
    • Henning Stahlberg
  4. Swiss Institute of Bioinformatics, Lausanne, Switzerland.

    • Matteo Dal Peraro
  5. Present addresses: Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford, UK (M.T.D.) and Institute of Anatomy, University of Bern, Bern, Switzerland (I.I.).

    • Matteo T Degiacomi &
    • Ioan Iacovache


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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Supplementary information


  1. Video 1: Aerolysin pore formation via a swirling mechanism (35.95 MB, Download)
    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.

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  1. Supplementary Text and Figures (1,766 KB)

    Supplementary Results, Supplementary Table 1 and Supplementary Figures 1–9.

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