Hemolysin E (HlyE) is a novel pore-forming toxin of Escherichia coli, Salmonella typhi, and Shigella flexneri. Here we report the X-ray crystal structure of the water-soluble form of E. coli HlyE at 2.0 A resolution and the visualization of the lipid-associated form of the toxin in projection at low resolution by electron microscopy. The crystal structure reveals HlyE to be the first member of a new family of toxin structures, consisting of an elaborated helical bundle some 100 A long. The electron micrographs show how HlyE oligomerizes in the presence of lipid to form transmembrane pores. Taken together, the data from these two structural techniques allow us to propose a simple model for the structure of the pore and for membrane interaction.

The novel pore-forming toxin hemolysin E (HlyE, ClyA, or SheA) consists of a long four-helix bundle with a subdomain (␤ tongue) that interacts with target membranes at one pole and an additional helix (␣ G ) that, with the four long helices, forms a five-helix bundle (tail domain) at the other pole. Random amino acid substitutions that impair hemolytic activity were clustered mostly, but not exclusively, within the tail domain, specifically amino acids within, adjacent to, or interacting with ␣ G . Deletion of amino acids downstream of ␣ G did not affect activity, but deletions encompassing ␣ G yielded insoluble and inactive proteins. In the periplasm Cys-285 (␣ G ) is linked to Cys-87 (␣ B ) of the four-helix bundle via an intramolecular disulfide. Oxidized HlyE did not form spontaneously in vitro but could be generated by addition of Cu(II) or mimicked by treatment with Hg(II) salts to yield inactive proteins. Such treatments did not affect binding to target membranes nor assembly into non-covalently linked octameric complexes once associated with a membrane. However, gel filtration analyses suggested that immobilizing ␣ G inhibits oligomerization in solution. Thus once associated with a membrane, immobilizing ␣ G inhibits HlyE activity at a late stage of pore formation, whereas in solution it prevents aggregation and consequent inactivation.

No abstract