(2013) 'Molecular assembly of the aerolysin pore reveals a swirling membrane-insertion mechanism.', Nature chemical biology., 9 (10). pp. 623-629. Further information on publisher's website:https://doi.org/10.1038/nchembio.1312Publisher's copyright statement:Additional information: Use policyThe full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-prot purposes provided that:• a full bibliographic reference is made to the original source • a link is made to the metadata record in DRO • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders.Please consult the full DRO policy for further details. AbstractAerolysin is the founding member of a super-family of ß-pore forming toxins for which the pore structure is unknown. We have combined X-ray crystallography, cryo-electron microscopy (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 utilized 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 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 ß-barrel.3

Organisms from all kingdoms produce pore-forming proteins, with the best-characterized being of bacterial origin. The last decade of research has revealed that the channels formed by these proteins can be very diverse, thus differentially affecting target cell-membrane permeability and consequent cellular outcome. The responses to these toxins are also extremely diverse due to multiple downstream effects of pore-induced changes in ion balance. Determining the secondary effects of pore-forming toxins is essential to understand their contribution to infection.

Owing to their pathogenical role and unique ability to exist both as soluble proteins and transmembrane complexes, pore-forming toxins (PFTs) have been a focus of microbiologists and structural biologists for decades. PFTs are generally secreted as water-soluble monomers and subsequently bind the membrane of target cells. Then, they assemble into circular oligomers, which undergo conformational changes that allow membrane insertion leading to pore formation and potentially cell death. Aerolysin, produced by the human pathogen Aeromonas hydrophila, is the founding member of a major PFT family found throughout all kingdoms of life. We report cryo-electron microscopy structures of three conformational intermediates and of the final aerolysin pore, jointly providing insight into the conformational changes that allow pore formation. Moreover, the structures reveal a protein fold consisting of two concentric β-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry.