title: Interaction of Escherichia coli hemolysin with biological membranes. A study using cysteine scanning mutagenesis.
abstract: Escherichia coli hemolysin (HlyA) is a membrane-permeabilizing protein belonging to the family of RTX-toxins. Lytic activity depends on binding of Ca2(+) to the C-terminus of the molecule. The N-terminus of HlyA harbors hydrophobic sequences that are believed to constitute the membrane-inserting domain. In this study, 13 HlyA cysteine-replacement mutants were constructed and labeled with the polarity-sensitive fluorescent probe 6-bromoacetyl-2-dimethylaminonaphthalene (badan). The fluorescence emission of the label was examined in soluble and membrane-bound toxin. Binding effected a major blue shift in the emission of six residues within the N-terminal hydrophobic domain, indicating insertion of this domain into the lipid bilayer. The emission shifts occurred both in the presence and absence of Ca2(+), suggesting that Ca2(+) is not required for the toxin to enter membranes. However, binding of Ca2(+) to HlyA in solution effected conformational changes in both the C-terminal and N-terminal domain that paralleled activation. Our data indicate that binding of Ca2(+) to the toxin in solution effects a conformational change that is relayed to the N-terminal domain, rendering it capable of adopting the structure of a functional pore upon membrane binding.
authors.affiliation: Institut für Medizinische Mikrobiologie und Hygiene, Universität Mainz, Germany. Schindel@mail.uni-mainz.de
authors.forename: C
authors.initials: C
authors.lastname: Schindel
authors.forename: A
authors.initials: A
authors.lastname: Zitzer
authors.forename: B
authors.initials: B
authors.lastname: Schulte
authors.forename: A
authors.initials: A
authors.lastname: Gerhards
authors.forename: P
authors.initials: P
authors.lastname: Stanley
authors.forename: C
authors.initials: C
authors.lastname: Hughes
authors.forename: V
authors.initials: V
authors.lastname: Koronakis
authors.forename: S
authors.initials: S
authors.lastname: Bhakdi
authors.forename: M
authors.initials: M
authors.lastname: Palmer
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes / Q000494:pharmacology; D006460:Hemolysin Proteins / Q000378:metabolism*; D006461:Hemolysis; D008081:Liposomes / Q000378:metabolism; D016296:Mutagenesis; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence
chemical_list: C122155:6-bromoacetyl-2-dimethylaminonaphthalene; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D008081:Liposomes; D015081:2-Naphthylamine; D003545:Cysteine; D002118:Calcium