Topography of Diphtheria Toxin A Chain Inserted into Lipid Vesicles

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Abstract
The membrane-inserting T domain of diphtheria toxin aids the low-pH-triggered translocation of the catalytic A chain of the toxin across endosomal membranes. To evaluate the role of the isolated A chain in translocation, the topography of isolated A chain inserted into model membrane vesicles was investigated using a mixture either of dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG) or of dimyristoleoylphosphatidylcholine (DMoPC) and DOPG. The latter mixture was previously found to promote deep insertion of the T domain. A series of single Cys mutants along the A chain sequence were labeled with bimane or BODIPY groups. After A chain insertion into model membranes, the location of these groups within the lipid bilayer was determined via bimane fluorescence emission lambda(max), binding of externally added anti-BODIPY antibodies, and a novel technique involving the comparison of the quenching of bimane fluorescence by aqueous iodide and membrane-associated 10-doxylnonadecane. The results show that in both DOPC- and DMoPC-containing bilayers, membrane-inserted residues all along the A chain sequence occupy shallow locations that are relatively exposed to the external solution. There were only small differences between A chain topography in the two different types of lipid mixtures. However, the behavior of the A chain in the two different lipid mixtures was distinct in that it strongly oligomerized in DMoPC-containing vesicles as judged by Trp fluorescence. In addition, A chain selectively induced fusion of the DMoPC-containing vesicles, and this may aid oligomerization by increasing the A chain/vesicle ratio. Fusion may also explain why A chain also selectively induced leakage of the contents of DMoPC-containing vesicles. We propose that isolated A chain is unlikely to be inserted in a transmembrane orientation, and thus its interaction with the T domain is likely to be critical for properly orienting the A chain within the bilayer in a fashion that allows translocation.

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