The overall results imply that the membrane interactions of the T-domain are critical in ensuring the proper conformational changes required for the preparation of the diphtheria toxin for the cellular entry. This contrasts with the evidence from the new and previously published data, obtained by spectroscopic measurements and molecular dynamics computer simulations, which indicate the refolding of TH1 upon the acidification of the isolated T-domain. Surprisingly, the TH1 helix maintains its conformation in the crystal of the full-length toxin even at pH 5. We demonstrate that neither catalytic nor receptor-binding domains change their structure upon this acidification, while the T-domain undergoes a conformational change that results in the unfolding of the TH2-3 helices. For the first time, we present the high-resolution structure of the diphtheria toxin at a mildly acidic pH (5-6) and compare it to the structure at neutral pH (7). Here, we use X-ray crystallography along with circular dichroism and fluorescence spectroscopy to gain insight into the mechanism of the early stages of pH-dependent conformational transition. The subsequent endosomal acidification triggers a series of conformational changes, resulting in the refolding and membrane insertion of the translocation (T-)domain and ultimately leading to the translocation of the catalytic domain into the cytoplasm. When pH trans is close to that of the cytosol, the N terminus is predominantly on the trans-side.ĭiphtheria toxin, an exotoxin secreted by Corynebacterium that causes disease in humans by inhibiting protein synthesis, enters the cell via receptor-mediated endocytosis. As illustrated on the right side, when the pH is the same on both sides of the membrane the N terminus is equally distributed. For K H, the value found for the second transition detected in the presence of membranes is used, pK H 5.7. The value of pH cis is fixed at 5.5, and the proportion of translocated N terminus, y, as a function of pH trans, is calculated with the following equation: y (K H /H t 1)/ (K H /H t K H /H c 2). Effect of a pH gradient on the proportion of the N terminus of T translocated on the trans-side of the membrane. The depths of the membrane hydrophobic core (HC) and the interface and the relative size of the-helix are taken from White and Wimley (7). The surface in black at pH 4 is neutral because of the protonation of acid amino acid side chains. One layer of the membrane is represented. Illustration of the localization of TH1 (end view) in the membrane interface before (pH 6) and after (pH 4) the second transition. With membranes of the translocation domain are finely tuned by pH changes to take advantage of the cellular uptake system. Our study shows how the structural changes and the interaction It arises from the changes in the balance of electrostatic attractionsĪnd repulsions between the N-terminal part and the membrane. The second step,Īs the pH decreases, leads to the functional inserted state. This is because of the pH-induced formation of a molten globule specialized for binding to the membrane.Īccumulation of this molten globule follows a precise molecular mechanism adapted to the toxin function. The first step involves hydrophobic interactionsīy the C-terminal region. In a sequential manner between the domain and the membrane during the insertion. We found that hydrophobic and electrostatic interactions occur During cell intoxication, this domain undergoes a change from a soluble and folded state atĪlkaline pH to a functional membrane-inserted state at acid pH. The study of the membrane insertion of the translocation domain of diphtheria toxin deepens our insight into the interactionsīetween proteins and membranes.
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