No 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.

DsbA, a member of the thioredoxin family of disulfide oxidoreductases, acts in catalyzing disulfide bond formation by donating its disulfide to newly translocated proteins. We have found that the two central residues within the active site Cys-30-Pro-31-His-32-Cys-33 motif are critical in determining the exceptional oxidizing power of DsbA. Mutations that change these two residues can alter the equilibrium oxidation potential of DsbA by more than 1000-fold. A quantitative explanation for the very high redox potential of DsbA was found by measuring the pKa of a single residue, Cys-30. The pKa of Cys-30 varied dramatically from mutant to mutant and could accurately predict the oxidizing power of each DsbA mutant protein.