The structure of a cytolytic α-helical toxin pore reveals its assembly mechanism

Abstract

Pore-forming toxins (PFTs) are a class of potent virulence factors that convert from a soluble form to a membrane-integrated pore. They exhibit their toxic effect either by destruction of the membrane permeability barrier or by delivery of toxic components through the pores. Among the group of bacterial PFTs are some of the most dangerous toxins, such as diphtheria and anthrax toxin. Examples of eukaryotic PFTs are perforin and the membrane-attack complex, proteins of the immune system. PFTs can be subdivided into two classes, alpha-PFTs and beta-PFTs, depending on the suspected mode of membrane integration, either by alpha-helical or beta-sheet elements. The only high-resolution structure of a transmembrane PFT pore is available for a beta-PFT--alpha-haemolysin from Staphylococcus aureus. Cytolysin A (ClyA, also known as HlyE), an alpha-PFT, is a cytolytic -helical toxin responsible for the haemolytic phenotype of several Escherichia coli and Salmonella enterica strains. ClyA is cytotoxic towards cultured mammalian cells, induces apoptosis of macrophages and promotes tissue pervasion. Electron microscopic reconstructions demonstrated that the soluble monomer of ClyA must undergo large conformational changes to form the transmembrane pore. Here we report the 3.3 A crystal structure of the 400 kDa dodecameric transmembrane pore formed by ClyA. The tertiary structure of ClyA protomers in the pore is substantially different from that in the soluble monomer. The conversion involves more than half of all residues. It results in large rearrangements, up to 140 A, of parts of the monomer, reorganization of the hydrophobic core, and transitions of -sheets and loop regions to -helices. The large extent of interdependent conformational changes indicates a sequential mechanism for membrane insertion and pore formation.

Mosquito Resistance to Bacterial Larvicidal Toxins

“…Other studies with the bacterial pneumolysin toxin have shown that there is indeed a conformational transition from the membrane-bound prepore to the transmembrane functional pore by the substantial refolding of -helical regions into membrane-insertedhairpins (Tilley Cell 2005) [56]. Very recently, the structural transformations of -sheets and loop regions to -helices have also been observed during pore formation of the Escherichia coli cytotoxin (ClyA) (Mueller Nature 2009) [57]. This may conceivably reflect that -helix 7 in the Cry4Ba pore-forming domain might be able to refold into an extended -hairpin to insert into the lipid membrane, though the hairpin contains several charged and polar-uncharged residues that are energetically unfavourable for membrane insertion.…”

Infection strategies of enteric pathogenic Escherichia coli

“…include ShET1 (characterized as an AB 5 toxin in Shigella flexneri) 62 and HlyE (a pore-forming toxin) 63 have also been proposed to contribute to EAEC virulence.…”

Membrane Damage by an α-Helical Pore-forming Protein, Equinatoxin II, Proceeds through a Succession of Ordered Steps

“…It was proposed that the observed structure is the prepore of actinoporins. Prepores were observed in many ␤-PFT (4) and were proposed also for some ␣-PFT (5,29).…”

“…In general, the details of the pore-forming mechanism of ␣-PFT are not as well understood as those of the ␤-PFT. Apart from structural details of the pore formed by cytolysin A from Escherichia coli (5), molecular properties of ␣-PFT pores are poorly known (i.e. the stoichiometry of the pore, protein regions that participate in the final pore, etc.)…”