Conformational Changes in Cholera Toxin B Subunit−Ganglioside GM1 Complexes Are Elicited by Environmental pH and Evoke Changes in Membrane Structure

Abstract

Fluorescence resonance energy transfer (FRET) was used to monitor pH-dependent structural changes in the cholera toxin B subunit (CTB) and the membranes with which CTB associates. The distance separating the single tryptophan (Trp88) of each CTB monomer and a pyrene probe linked to the membrane-imbedded tail of ganglioside GM1 is not influenced by pH in a range from 3.5 to 7.5, consistent with the position of Trp88 in the GM1 binding site of CTB. In contrast, the distance between the pyrene probe on GM1 and coumarin, stilbene, or fluorescein probes covalently linked to specific sites on CTB appears to increase significantly as the pH is lowered to 5.0 or less. This conformational change is not accompanied by detectable changes in the distance between Trp88 and these extrinsic probe positions in the presence of nonfluorescent GM1. However, when the distance from Trp88 to the extrinsic probes is monitored as a function of pH in the absence of GM1, a conformational change is seen which indicates that receptor binding influences the character of pH-dependent conformational changes that occur within CTB. Interestingly, the observed change in CTB conformation is accompanied by a change in the relative position of GM1 within the membrane as judged by FRET from the pyrene probe on GM1 to a 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) probe linked to the polar head group of phosphatidylethanolamine and positioned at the membrane surface. Taken together, the data imply that low endosomal pH is capable of inducing structural changes in CTB, which, in turn, exert effects on the structure of the membrane to which CTB is bound. These phenomena may have a role in (1) processing of cholera toxin within the endosomal compartments of some target cell types, (2) determining the lag time between cholera toxin binding and the target cell response to cholera intoxication, or (3) the efficiency of CTB and cholera toxin as mucosal adjuvants.

Processing of Pseudomonas aeruginosa Exotoxin A Is Dispensable for Cell Intoxication

“…Indeed, the ER-targeted toxins Shiga toxin and cholera toxin are closely related AB5 toxins that recognize glycolipids. Once in the endosome lumen, low pH induces in these toxins only subtle conformational changes that are unrelated to membrane penetration (29,31). These toxins eventually reach the ER, where they can be visualized (27,52).…”

Differential targeting of secretory lysosomes and recycling endosomes in mast cells revealed by patterned antigen arrays

“…Furthermore, these results indicate that the slow mobility of the bound CTB cannot be ascribed simply to an increased surface level of CTB-GM1 as a result of stimulated outward trafficking. Slow lateral diffusion of CTB-GM1 has been observed in other cell types (Bacia et al, 2002;Kenworthy et al, 2004), and its physical basis remains to be determined, although it has been suggested to be pH dependent (McCann et al, 1997;Pelkmans et al, 2004). In any case, this slow lateral mobility causes freshly delivered CTB-GM1 to accumulate at a targeting site that corresponds to the clustered IgE receptors and initial stimulus.…”

Mutational Analysis of Ganglioside GM1-Binding Ability, Pentamer Formation, and Epitopes of Cholera Toxin B (CTB) Subunits and CTB/Heat-Labile Enterotoxin B Subunit Chimeras

“…Apparently, the loss of the hydrogen bonds between the terminal galactose and either Glu-51 or Lys-91 does not dramatically affect GM 1 binding in vitro. The interactions between Glu-51 and Lys-91, together with His-57 (all conserved between CT and LT-I), have been proposed in LT-I to be involved in a pH-dependent conformational change of the pentamer (33), a change that also occurs with CTB (22,33). Efforts in those studies to make site-directed mutations for Glu-51 and Lys-91 in the B subunit of LT-I were unsuccessful, leading to the suggestion that these mutations were toxic to the E. coli host.…”