Insertion of Escherichia coli alpha-haemolysin in lipid bilayers as a non-transmembrane integral protein: prediction and experiment.

alpha-Haemolysin is an extracellular protein toxin (approximately 107 kDa) secreted by Escherichia coli that acts at the level of the plasma membranes of target eukaryotic cells. The nature of the toxin interaction with the membrane is not known at present, although it has been established that receptor-mediated binding is not essential. In this work, we have studied the perturbation produced by purified alpha-haemolysin on pure phosphatidylcholine bilayers in the form of large unilamellar vesicles, under conditions in which the toxin has been shown to induce vesicle leakage. The bilayer systems containing bound protein have been examined by differential scanning calorimetry, fluorescence spectroscopy, differential solubilization by Triton X-114, and freeze-fracture electron microscopy. All the data concur in indicating that alpha-haemolysin, under conditions leading to cell lysis, becomes inserted in the target membrane in the way of intrinsic or integral proteins. In addition, the experimental results support the idea that inserted alpha-haemolysin occupies only one of the membrane phospholipid monolayers, i.e. it is not a transmembrane protein. The experimental data are complemented by structure prediction studies according to which as many as ten amphipathic alpha-helices, appropriate for protein-lipid interaction, but no hydrophobic transmembrane helices are predicted in alpha-haemolysin. These observations and predictions have important consequences for the mechanism of cell lysis by alpha-haemolysin; in particular, a non-transmembrane arrangement of the toxin in the target membrane is not compatible with the concept of alpha-haemolysin as a pore-forming toxin.

Reversible denaturation, self-aggregation, and membrane activity of Escherichia coli alpha-hemolysin, a protein stable in 6 M urea.

Escherichia coli alpha-hemolysin (HlyA) is an extracellular protein toxin (107 kDa) whose cell lytic activity may be preserved for months at -20 degreesC in the presence of 6 M urea, although it decays rapidly in urea-free buffers. This paper describes experiments addressed to unravel the role of urea in HlyA stabilization. Urea up to 8 M inhibits the Ca2+-binding and hemolytic activities of the protein, alters its secondary and tertiary structures, and reduces its tendency to self-aggregation. All these changes are largely reversed upon urea removal by dilution or dialysis, suggesting that they are interrelated. Furthermore, the extent of recovery of the native activities and structural features of alpha-hemolysin that follows urea removal increases with the concentration of urea during the previous phase. Thus, it seems that urea elicits the reversible transition of HlyA to a less active but more stable state whose structure differs significantly from that of the native protein. Moreover dialysis equilibration of the protein with buffers containing 3 M urea induces the formation of a molecular form of HlyA 5-10 times more active than the native protein in the absence of urea. This hyperactive intermediate appears to keep the native secondary structure of HlyA, but with a less compact tertiary structure, that increases the number of exchangeable Ca2+ ions under these conditions. Changes in the intrinsic fluorescence of HlyA also support the notion of a conformational change in the high-activity intermediate. The intermediate is only detected when assayed in the presence of Ca2+ and 3 M urea and can bind a large number of calcium ions (approximately 12 vs approximately 3 for the native protein); it shows a large tendency to self-aggregation and presumably, in the presence of membranes, a similar tendency to irreversible insertion, which may be the reason for its high lytic activity.

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

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.

Purification of Escherichia coli pro-haemolysin, and a comparison with the properties of mature alpha-haemolysin.

Pro-haemolysin (approximately 110 kDa), the inactive precursor of the membrane-lytic toxin alpha-haemolysin, has been purified from an overproducing strain of Escherichia coli. Pro-haemolysin forms aggregates in aqueous media, like the mature protein, suggesting an amphipathic structure. Direct measurements of protein binding to liposomal membranes, following a novel procedure, show that pro-haemolysin can bind the lipid bilayers to a similar extent as alpha-haemolysin. This is confirmed by the observed changes in the intrinsic fluorescence emission of the protein upon binding the bilayers. However, pro-haemolysin is totally unable to induce liposomal membrane lysis. Binding of Ca2+, that is essential for the lytic activity of alpha-haemolysin, is greatly diminished in the precursor protein, as shown both by direct measurements of 45Ca(2+)-binding and by fluorescence measurements. The results suggest that binding of a fatty acyl residue in the activation step brings about an important conformational change in the protein that involves the Ca(2+)-binding domain.

The binding of divalent cations to Escherichia coli alpha-haemolysin.

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+.