Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process

Abstract

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.

A General Strategy of Aerolysin Nanopore Detection for Oligonucleotides with the Secondary Structure

“…The secondary structure is pervasive in oligonucleotides, which is essential for constituting the functional domain toward the binding and recognition. Due to the extremely narrow diameter (≈1 nm), the aerolysin only offers the possibility to squeeze a linear oligonucleotide into its lumen, however, it cannot accommodate a structured oligonucleotide with large volume. Compared with the widely applied α‐hemolysin, the aerolysin lacks a wide vestibule which permits the unfolding of a structured oligonucleotide for the successive translocation through the narrowest constriction.…”

Identification and characterization of virulent Aeromonas hydrophila Ah17 from infected Channa striata in river Cauvery and in vitro evaluation of shrimp chitosan

“…They are the founding members of a large superfamily (β‐PTFs) that span all the kingdom of life (Szczesny et al, ). Studies on cryo‐electron microscopy showed that the bacterial PFTs are generally secreted as water soluble monomers and binds with target membranes and assemble into the circular oligomers, which undergoes the conformational changes that allow membrane insertion leading to pore formation and finally potential cell death (Iacovache et al, ). In the present study, the strain Ah17 harbors both aerolysin ( aer ) and hemolysin ( hly ) genes and the presence of these genes evidently supports the pathogenic nature of A. hydrophila Ah17.…”

The aerolysin nanopore: from peptidomic to genomic applications

“…1a and b). 19,20 Molecular dynamics simulations on a 10 2 ns timescale were stable for both wild-type and Y221G mutant pro-aeroly-sins. After refinement using a molecular dynamics flexible fitting procedure, the best heptameric model had a cross-correlation coefficient with the cryo-EM map of 0.79.…”

“…The aerolysin prepore goes through several conformational changes like initial rearrangements, oligomerization, the concentric barrel fold, tightly bound together by hydrophobic interactions, and then forms the final ARP with a β-barrel length of ~87 Å and a diameter of ~8.75 Å (average distance between the opposite strand backbone). 19 Structurally, the ARP has multiple charged residues in its channel wall, particularly around both openings to the pore. Negatively charged residues include E237, E252, E254, and E258 near the trans ARP entrance, and D209, D216 near the cis entrance, while positive charges contributed by K238, K242, K244, and K246 localize towards the trans barrel, while charges from R220, R282, and R288 cluster around the cis entrance.…”

“…These studies suggested an ultrastability of ARP and its stoichiometry. 19,20 Compared to other reported biological nano-pores, ARP exhibits the advantage of stability over a broad pH range. 21,22 …”