Use of Trp Mutations To Evaluate the Conformational Behavior and Membrane Insertion of A and B Chains in Whole Diphtheria Toxin

Abstract

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.

Topography of Helices 5–7 in Membrane-inserted Diphtheria Toxin T Domain

“…Last, helices 6 and 7 are separated by a kink formed by a Pro-Gly sequence, which is somewhat unfavorable for transmembrane insertion. 2 In previous studies, we found that the natural Trp in the center of helix 5 inserted into lipid bilayers (21,22). In a recent paper, Senzel et al (23) demonstrated that, at least in the state in which the T domain has an open pore, residues at the N-terminal of helix 5, along with the remainder of the Nterminal half of the T domain, can reach the trans side of the bilayer (i.e.…”

Ligand-mediated conformational changes and positioning of tryptophans in reconstituted human sodium/d-glucose cotransporter1 (hSGLT1) probed by tryptophan fluorescence

“…Tryptophan residues have been successfully used as fluorescent probes to investigate conformational changes in membrane proteins [6,7,12,14,[22][23][24][25][26][27][28][29][30]. Our recent success in functional hSGLT1 expression and purification from P. pastoris and identification of different conformational states of hSGLT1 in solution [7] raised the question whether these conformational changes can also be observed in proteoliposomes in a reconstituted form, an experimental condition closer to the in vivo situation of membrane proteins.…”

Diphtheria toxin

“…As pH decreases from 7 to 4 (or below), the C domain has also a tendency to partially unfold, adopt a molten, globulelike state, and, as a result, bind and penetrate negatively charged phospholipid bilayers (Figure 4.6) [52,131]. However, these transitions occur at lower pH values and never reach full completion, as compared to those adopted by the T domain.…”

“…The role of the T domain is to assist the C domain in these transitions (Figure 4.6). Under acid conditions, the T domain in solution, as well as bound or inserted into a lipid membrane, has the propensity to bind proteins in a molten globule state [52,131,132]. More precisely, the T domain stabilizes the partially folded states of the C domain corresponding to each step of the process [52].…”

“…These transitions of C (partial unfolding, membrane binding, and membrane penetration) occur at a higher pH of about 1 pH unit than in the absence of T, and they also occur to a fuller extent (Figure 4.6). In addition, contacts between the two domains significantly reduce the exposure of the C domain to lipids [131,133]. Thus, the T domain acts as a specialized pH-dependent chaperone for the C domain.…”