Interaction of diphtheria toxin with model membranes

Abstract

Low pH is believed to trigger membrane penetration by diphtheria toxin in vivo. The efefct of pH upon the binding of the toxin to unilamellar model membrane vesicles was determined by using a fluorescence quenching assay. A series of studies were undertaken to determine the effect of lipid composition upon the binding of lipids to the toxin. The binding of toxin to various small unilamellar vesicles of zwitterionic or anionic lipids was similar in extent and was accompanied by deep penetration of the toxin into the fatty acyl chains, in agreement with previous studies. However, the transition pH, which is the pH at and below which toxin binding becomes significant, depended upon the fraction of anionic lipids, being highest with model membranes composed totally of anionic lipids (pH 5.8) and lowest with membranes composed of zwitterionic lipids (pH 5.2). Except for vesicle charge, the transition pH was independent of the nature of the lipid polar groups used. High ionic strength, which had no effect on the transition pH with zwitterionic vesicles, was found to shift the transition pH with totally anionic vesicles to pH 5.2. This suggests that both direct protein-lipid electrostatic interactions and the ionic double layer, which gives rise to a low local pH around anionic vesicles, contribute to the shift in the transition pH. The effect of lipid composition upon the kinetics and strength of binding was also examined. At low pH, binding was rapid and tight. Binding to vesicles containing 20 wt % anionic phosphatidylglycerol was faster and tighter than binding to vesicles of zwitterionic phosphatidylcholine. In addition, binding to phosphatidylcholine vesicles was decreased markedly both when vesicle size was large and when toxin was preincubated in the absence of lipid, whereas these behaviors were not observed with vesicles containing 20% phosphatidylglycerol. These differences probably arise from electrostatic interactions as well. We conclude that the interaction of toxin with lipid vesicles at low pH has both hydrophobic and electrostatic components. This has important implications for both the behavior of the toxin in vivo and the design of experiments for study of toxin behavior in model membranes.