Calcium-dependent conformation of E. coli α-haemolysin. Implications for the mechanism of membrane insertion and lysis

Abstract

Previous studies from this laboratory had shown that calcium ions were essential for the membrane lytic activity of E. coli alpha-haemolysin (HlyA), while zinc ions did not sustain such a lytic activity. The present data indicate that calcium-binding does not lead to major changes in the secondary structure, judging from circular dichroism spectra. However binding to Ca2+ exposes new hydrophobic residues at the protein surface, as indicated by the increased binding of the fluorescent probe aniline naphtholsulphonate (ANS), and by the increased tendency of the Ca2+-bound protein to self-aggregate. In addition zinc ions are seen to decrease the thermal stability of HlyA which, according to intrinsic fluorescence and differential scanning calorimetry data, is stable below 95 degrees C when bound to calcium, while it undergoes irreversible denaturation above 60 degrees C in the zinc-bound form. Binding to phosphatidylcholine bilayers is quantitatively similar in the presence of both cations, but about one-third of the zinc-bound HlyA is released in the presence of 2 M NaCl. Differential scanning calorimetry of dimyristoylglycerophosphocholine large unilamellar vesicles reveals that Zn2+-HlyA interaction with the lipid bilayer has a strong polar component, while Ca2+-HlyA appears to interact mainly through hydrophobic forces. Experiments in which HIyA transfer is measured from phospholipid vesicles to red blood cells demonstrate that Ca2+ ions promote the irreversible binding of the toxin to bilayers. All these data can be interpreted in terms of a specific Ca2+ effect that increases the surface hydrophobicity of the protein, thus facilitating its irreversible bilayer insertion in the fashion of intrinsic membrane proteins.

E. coli <FONT FACE=Symbol>a</FONT>-hemolysin: a membrane-active protein toxin

“…Our previous studies (66) have shown the difference between adsorption and insertion, and the requirement of a fluid (liquid disordered) bilayer for irreversible insertion. The more recent data by Bakás et al (84) demonstrate that Ca 2+ -binding is an additional requirement for irreversible insertion. This kind of insertion appears to be essential for the lytic process to go on (through yet unknown steps).…”

“…This suggests that Ca 2+ induces in the protein in solution a state such that the toxin becomes capable of producing membrane lysis. The same liposome-red blood cell competition assay for the irreversibility of Hly binding was applied by Bakás et al (84) in a new series of experiments to study the effects of Ca 2+ on toxin binding irreversibility and subsequent membrane damage, with the result that Ca 2+ facilitates the irreversible bilayer insertion of the protein. Looking in more detail into the HlyAcalcium system, the above authors observed that, even in the absence of lipid bilayers, Ca 2+ binding had significant effects on α-hemolysin.…”

“…It should be noted that Ca 2+ binding does not elicit significant changes in the protein secondary structure, judging from circular dichroism measurements (84). However, other techniques, namely studies of thermally induced change of intrinsic fluorescence, and trypsin digestion under controlled conditions, are compatible with a Ca 2+ -induced change in conformation (84).…”

“…However, other techniques, namely studies of thermally induced change of intrinsic fluorescence, and trypsin digestion under controlled conditions, are compatible with a Ca 2+ -induced change in conformation (84). Calcium ions would then induce a change in tertiary structure that would result in surfacing hydrophobic portions of the protein.…”

“…Thus the requirement of Ca 2+ ions for HlyA-dependent hemolysis refers at least to the requirement of Ca 2+ for a proper (irreversible) insertion into the membrane. Such an insertion, that converts the toxin into an intrinsic membrane protein (84), is undoubtedly favored by the calcium-dependent change in tertiary structure leading to the exposure of hydrophobic patches. Calcium ions may be playing in α-hemolysin or in the B. pertussis toxin (87) the role of pH or temperature in the pre-insertion conforma- tional changes that are observed in colicin A (49) or in Pseudomonas exotoxin A (54).…”