The repeat domain of Escherichia coli haemolysin (HlyA) is responsible for its Ca2+-dependent binding to erythrocytes.
The haemolysin protein (HlyA) of Escherichia coli contains 11 tandemly repeated sequences consisting of 9 amino acids each between amino acids 739 and 849 of HlyA. We removed, by oligonucleotide-directed mutagenesis, different single repeats and combinations of several repeats. The resulting mutant proteins were perfectly stable in E. coli and were secreted with the same efficiency as the wild-type HlyA. HlyA proteins which had lost a single repeat only were still haemolytically active (in the presence of HlyC) but required elevated levels of Ca2+ for activity, as compared to the wild-type haemolysin. Removal of three or more repeats led to the complete loss of the haemolytic activity even in the presence of high Ca2+ concentrations. The mutant haemolysins were unable to compete with the wild-type haemolysin for binding to erythrocytes at low Ca2+ concentrations but could still generate ion-permeable channels in artificial lipid bilayer membranes formed of plant asolectin, even in the complete absence of Ca2+. These data indicate that the repeat domain of haemolysin is responsible for Ca2+-dependent binding of haemolysin to the erythrocyte membrane. A model for the possible functional role of Ca2+ in haemolysis is presented.
Ludwig
A
A
Institut für Genetik und Mikrobiologie, Universität Würzburg, Federal Republic of Germany.
Jarchau
T
T
Benz
R
R
Goebel
W
W
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive; D002118:Calcium / Q000502:physiology; D004910:Erythrocyte Membrane / Q000382:microbiology*; D004926:Escherichia coli / Q000235:genetics / Q000472:pathogenicity*; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism; D008954:Models, Biological; D009153:Mutagens; D011485:Protein Binding; D011487:Protein Conformation; D015183:Restriction Mapping
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D008051:Lipid Bilayers; D009153:Mutagens; D002118:Calcium