title: The binding of divalent cations to Escherichia coli alpha-haemolysin.
abstract: alpha-haemolysin, an extracellular protein toxin of Escherichia coli, is known to disrupt eukaryotic cell membranes. In spite of genetic evidence of Ca(2+)-binding motifs in its sequence, conflicting results are found in the literature on the requirement of divalent cations for the membranolytic activity of the toxin. Moreover, Ca(2+)-binding sites have not been characterized to date in the native protein. The results in this paper show that when Ca2+ levels are kept sufficiently low during bacterial growth and toxin purification, membrane lysis does not occur in the absence of added divalent cations. Ca2+ and, at higher concentrations, Sr2+ and Ba2+, support the lytic activity, but Mg2+, Mn2+, Zn2+ and Cd2+ appear to be inactive in this respect. Binding of metal ions can be followed by changes in the intrinsic fluorescence of alpha-haemolysin; ions supporting lytic activity produce changes in the intrinsic fluorescence that are not caused by the inactive ones. Scatchard analysis of 45Ca2+ binding reveals three equivalent, independent sites, with Kd approximately 0.11 mM. No 45Ca2+ binding is observed when the protein is incubated with Zn2+; conversely, incubation with Ca2+ prevents subsequent binding of 65Zn2+. In the light of three-dimensional data available for a structurally related protein, alkaline protease of Pseudomonas aeruginosa [Baumann, U., Wu, S., Flaherty, K. M. & McKay, D. B. (1993) EMBO J. 12, 3357-3364] it is suggested that alpha-haemolysin may bind a larger number of Ca2+ than the three that are more easily exchangeable and are thus detected in the 45Ca(2+)-binding experiments. In addition, structural similarities and conservation of ion-binding motifs support the hypothesis that His 859 is involved in the mutually exclusive binding of Zn2+ and Ca2+.
authors.affiliation: Department of Biochemistry, University of the Basque Country, Bilbao, Spain.
authors.forename: H
authors.initials: H
authors.lastname: Ostolaza
authors.forename: A
authors.initials: A
authors.lastname: Soloaga
authors.forename: F M
authors.initials: FM
authors.lastname: Goñi
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005453:Fluorescence; D006460:Hemolysin Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D015032:Zinc / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D015032:Zinc; D002118:Calcium