title: Structure-function relationships of a novel bacterial toxin, hemolysin E. The role of alpha G.
abstract: The novel pore-forming toxin hemolysin E (HlyE, ClyA, or SheA) consists of a long four-helix bundle with a subdomain (beta tongue) that interacts with target membranes at one pole and an additional helix (alpha(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 alpha(G). Deletion of amino acids downstream of alpha(G) did not affect activity, but deletions encompassing alpha(G) yielded insoluble and inactive proteins. In the periplasm Cys-285 (alpha(G)) is linked to Cys-87 (alpha(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 alpha(G) inhibits oligomerization in solution. Thus once associated with a membrane, immobilizing alpha(G) inhibits HlyE activity at a late stage of pore formation, whereas in solution it prevents aggregation and consequent inactivation.
authors.affiliation: Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom.
authors.forename: A
authors.initials: A
authors.lastname: Atkins
authors.forename: N R
authors.initials: NR
authors.lastname: Wyborn
authors.forename: A J
authors.initials: AJ
authors.lastname: Wallace
authors.forename: T J
authors.initials: TJ
authors.lastname: Stillman
authors.forename: L K
authors.initials: LK
authors.lastname: Black
authors.forename: A B
authors.initials: AB
authors.lastname: Fielding
authors.forename: M
authors.initials: M
authors.lastname: Hisakado
authors.forename: P J
authors.initials: PJ
authors.lastname: Artymiuk
authors.forename: J
authors.initials: J
authors.lastname: Green
mesh_terms: D000595:Amino Acid Sequence; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000502:physiology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
chemical_list: D006460:Hemolysin Proteins