Use of Trp mutations to evaluate the conformational behavior and membrane insertion of A and B chains in whole diphtheria toxin

The structure of diphtheria toxin was examined using its Trp fluorescence. To examine the interactions of the A and B chains of the toxin independently, mutants were constructed in which Trp residues were restricted to either the A or the B chain. The conformation and stability of the mutants were very similar to those of the wild-type protein. In addition, they underwent the low-pH conformational transition and membrane insertion at about the same pH as wild-type toxin. This shows Trp do not play a critical role in these processes which are necessary for the translocation of toxin across endosomal membranes in vivo. There was a shift in fluorescence of the Trp mutants which showed the low-pH-induced transition increases exposure of both the A and B Trp to a more polar environment. This supports a model in which the interdomain interactions present at neutral pH break down at low pH. To evaluate the location of the A and B chains in the membrane, the fluorescence quenching of model membrane inserted toxin was measured. Comparison of the amount of quenching by lipid labeled with nitroxides localized at shallow, medium, or deep depths within the bilayer demonstrated that both the A and B chains insert deeply, but the A chain Trp are somewhat less deeply inserted. Trp on the A chain are also less exposed to lipid than on the B chain, as judged by their weaker quenching by the nitroxide-labeled lipid. This conclusion was supported by the observation that the Trp of membrane-inserted isolated A chain is more lipid-exposed than when the A chain is part of the whole toxin. Both the A and B chain Trp become less exposed to Lipid after neutralizing pH. However, both chains remain inserted, with at least part of the B chain remaining deeply inserted. These results support the "partial wrapper" model in which both the A and B chains are inserted but contacts between the two chains significantly reduce the exposure of the A chain to lipid.