Structure-function relationships of a novel bacterial toxin, hemolysin E -The role of alpha(G)

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 othe r pole. Random amino acid substitutions that-impair hemolytic activity were clustered mostly, but clot exclusively, within the tail domain, specifical ly-amino acids within, adjacent to, or interacting with cud. Deletion of am ino acids downstream of alpha (G) did not affect activity, but deletions en compassing alpha (G) yielded insoluble and inactive proteins. In the peripl asm Cys-285 (alpha (G)) is linked to Cys-87 (alpha (B)) of the four-helix b undle via an intramolecular disulfide, Oxidized HlyE did not form spontaneo usly in vitro but could be generated by addition of Cu(II) or mimicked by t reatment with Hg(II) salts to yield inactive proteins, Such treatments did not affect binding to target membranes:nor assembly into non-covalently lin ked octameric complexes once associated with a membrane. However, gel filtr ation analyses suggested that immobilizing alpha (G) inhibits oligomerizati on in solution. Thus once associated with a membrane, immobilizing alpha (G ) inhibits HlyE activity at a late stage of pore formation, whereas in solu tion it prevents aggregation and consequent inactivation.