The repeat domain of Escherichia coli haemolysin (HlyA) is responsible for its Ca2+-dependent binding to erythrocytes

Abstract

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.

The Calcium-binding C-terminal Domain of Escherichia coli α-Hemolysin Is a Major Determinant in the Surface-active Properties of the Protein

“…Various reports have linked the RTX domain to target membrane binding (13,14). In addition we had found that the sequence 914 -936, located very close to that domain, inter- acted specifically with ␣-glycophorin, an HlyA receptor in human erythrocytes (9,10).…”

“…We had also shown that a short sequence in the C-terminal domain (aa 914 -936) was the main HlyA region recognizing the ␣-glycophorin receptor (10), and earlier reports suggested that the nonapeptide repeat, calcium-binding region was involved in the early stages of receptor/membrane binding by HlyA (13,14). It was therefore decided to clone and express the nonapeptide repeat region, to study its properties in the absence of the amphipathic helix domain.…”

Chitinase B from Serratia marcescens BJL200 is exported to the periplasm without processing

“…Site-directed mutagenesis studies have indicated that negatively charged residues could be important (Stanley et al, 1991;Kenny e t al., 1992). A feature seen in most proteins secreted by a haemolysinlike mechanism is the presence of repeats of the sequence GGXGXD that are involved in calcium binding (Ludwig et al, 1988;Akatsuka et al, 1994). The relevance of these repeats for export is, however, unknown.…”

Oscillin, an extracellular, Ca2+‐binding glycoprotein essential for the gliding motility of cyanobacteria

“…Protein similarity searches revealed extensive similarity to a large family of secretory proteins, which includes the RTX-toxins, extracellular zinc proteases and various bacterial exoproteins (Ludwig et al, 1988). All these proteins contain a variable number of tandem repeats of a nine amino acid motif rich in glycine, aspartic acid and asparagine.…”