pH-Triggered Conformational Switching of the Diphtheria Toxin T-Domain: The Roles of N-Terminal Histidines

Abstract

pH-Induced conformational switching is essential for functioning of diphtheria toxin, which undergoes a membrane insertion/translocation transition triggered by endosomal acidification as a key step of cellular entry. In order to establish the sequence of molecular rearrangements and side chain protonation accompanying the formation of the membrane-competent state of the toxin’s translocation (T) domain, we have developed and applied an integrated approach that combines multiple techniques of computational chemistry (e.g., long, µsec-range, all-atom molecular dynamics simulations; continuum electrostatics calculations; and thermodynamic integration) with several experimental techniques of fluorescence spectroscopy. Thermodynamic integration calculations indicate that protonation of H257 causes the greatest destabilization of the native structure (6.9 kcal/mole), which is consistent with our early mutagenesis results. Extensive equilibrium MD simulations with a combined length of over eight µsec demonstrate that histidine protonation, while not accompanied by the loss of structural compactness of the T-domain, nevertheless results in substantial molecular rearrangements characterized by the partial loss of secondary structure due to unfolding of helices TH1 and TH2, and the loss of close contact between the C- and N-terminal segments. The structural changes accompanying the formation of the membrane-competent state ensure an easier exposure of the internal hydrophobic hairpin formed by helices TH8 and TH9, in preparation for its subsequent transmembrane insertion.

Hydrogen–Deuterium Exchange and Mass Spectrometry Reveal the pH-Dependent Conformational Changes of Diphtheria Toxin T Domain

“…15 At pH 5.5, the T domain further unfolds, resulting in the exposure of its initially buried C-terminal hydrophobic helices (TH8–9) and subsequent oligomerization.…”

Diphtheria toxin conformational switching at acidic pH

“…Experiments using the hydrophobic photoactivable reagent diamonofluorescein showed the TH1, TH8 and TH9 a-helices to be inserted into the hydrophobic core of the lipid bilayer [24]. A more comprehensive approach combining fluorescence spectroscopy with extensive molecular dynamics [30] suggests that the first step of the conformational transition is represented by the partial loss of the TH1 and TH2 helical structure, an event that allows the exposure of the hairpin formed by the TH8 and TH9 a-helices and their access to the membrane. At variance with this, the present structure indicates that the first portion of the T domain to unfold comprises the TH2, TH3 and partially the TH4 a-helices (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].…”

“…Extensive equilibrium molecular dynamic simulations suggest that His protonation results in substantial molecular rearrangements characterized by the partial loss of secondary structure due to the partial unfolding of helices TH1 and TH2 and the loss of close contact between the C-and N-terminal segments of the T domain [114]. The structural changes accompanying the formation of the membrane-competent state ensure easier exposure of the internal hydrophobic hairpin formed by helices TH8 and TH9, in preparation for its subsequent transmembrane insertion.…”