Conformational Switching of the Diphtheria Toxin T Domain

Abstract

The diphtheria toxin T-domain translocates the catalytic C-domain across the endosomal membrane in response to acidification. To elucidate the role of histidine protonation in modulating pH-dependent membrane action of the T-domain, we have used site-directed mutagenesis coupled with spectroscopic and physiological assays. Replacement of H257 with an arginine (but not with a glutamine) resulted in dramatic unfolding of the protein at neutral pH, accompanied by a substantial loss of helical structure and greatly increased exposure of the buried residues W206 and W281. This unfolding and spectral shift could be reversed by the interaction of the H257R mutant with model lipid membranes. Remarkably, this greatly unfolded mutant exhibited WT-like activity in channel formation, N-terminus translocation and cytotoxicity assays. Moreover, membrane permeabilization caused by H257R mutant occurs already at pH 6, where wild type protein is inactive. We conclude that protonation of H257 acts as a major component of the pH-dependent conformational switch, resulting in destabilization of the folded structure in solution and thereby promoting the initial membrane interactions necessary for translocation.

Membrane translocation assay based on proteolytic cleavage: Application to diphtheria toxin T domain

“…The results obtained for both POPG (red) and POPS (blue) clearly indicate that a certain fraction of anionic lipids is needed for translocation activity. This threshold behavior is consistent with our previous biophysical observations, indicating that anionic lipids decrease the energy barrier for the transmembrane insertion transition and that the T domain is kinetically trapped in the interfacial intermediate state at low anionic lipid content [15; 18]. This intermediate state is not functionally relevant and lacks the transmembrane orientation of the helices.…”

“…We demonstrate that the cleavage of the His-tag in the course of T domain interaction with thrombin-loaded vesicles occurs only when the N-terminal part had been translocated across the bilayer into the vesicle. We applied this method to studying dependence of LUV translocation efficiency on lipid composition of the artificial membrane; the results confirm our previous statement that a high content of acidic phospholipids is important for the formation of the final inserted state [15]. We have also studied the activity of several T domain mutants carrying replacements of certain histidine residues with neutral or positively charged ones, and results obtained are in good agreement with cell death assay data.…”

“…4B), reproduced from our previous publications [5; 15]. The cell death assay is performed using a weakened strain of the full-length protein and is based on monitoring the inhibition of protein synthesis [6].…”

“…It is likely that the leakage is caused during the transient initial binding event by the redistribution of the mass between the leaflets due to lipid flip-flop, and not by the formation of the stable pore. Therefore, while leakage studies can be very useful for detecting initial membrane interactions and for comparing various mutants and bilayer lipid compositions [5; 15], their results should always be verified with other more specific functional assays. For example, the leakage assay, when applied to histidine mutants of the T domain, does not demonstrate substantial differences from the WT protein, except for slightly slowed kinetics in the case of glutamine substitutions (Fig.…”

Diphtheria toxin

“…His223, His257, and His251 are the most sensitive triggers for the formation of the molten globule state in solution, whereas His322, His323, and His251 are the most sensitive triggers for membrane binding [112]. H257 causes the greatest destabilization of the native structure [113,114]. H322 found between the C-terminal helices TH8 and TH9 plays a role in channel formation and translocation of the N-terminal helices through the lipid bilayer [115,116].…”