Behavior of the N-Terminal Helices of the Diphtheria Toxin T Domain during the Successive Steps of Membrane Interaction

Abstract

During intoxication of a cell, the translocation (T) domain of the diphtheria toxin helps the passage of the catalytic domain across the membrane of the endosome into the cytoplasm. We have investigated the behavior of the N-terminal region of the T domain during the successive steps of its interaction with membranes at acidic pH using tryptophan fluorescence, its quenching by brominated lipids, and trypsin digestion. The change in the environment of this region was monitored using mutant W281F carrying a single native tryptophan at position 206 at the tip of helix TH1. The intrinsic propensity to interact with the membrane of each helix of the N-terminus of the T domain, TH1, TH2, TH3, and TH4, was also studied using synthetic peptides. We showed the N-terminal region of the T domain was not involved in the binding of the domain to the membrane, which occurred at pH 6 mainly through hydrophobic effects. At that stage of the interaction, the N-terminal region remained strongly solvated. Further acidification eliminated repulsive electrostatic interactions between this region and the membrane, allowing its penetration into the membrane by attractive electrostatic interactions and hydrophobic effects. The peptide study indicated the nature of forces contributing to membrane penetration. Overall, the data suggested that the acidic pH found in the endosome not only triggers the formation of the molten globule state of the T domain required for membrane interaction but also governs a progressive penetration of the N-terminal part of the T domain in the membrane. We propose that these physicochemical properties are necessary for the translocation of the catalytic domain.

Characterization of a Membrane-active Peptide from the Bordetella pertussis CyaA Toxin

“…We hypothesized that regions able to partition from solution to membrane should exhibit some canonical features like secondary structure propensity and amphiphilicity (79,80). Two regions with potential membrane-interacting properties were thus identified, located from residue 414 to 440 and from residue 454 to 484.…”

Concerted Protonation of Key Histidines Triggers Membrane Interaction of the Diphtheria Toxin T Domain

“…Protein expression was performed as described previously (15,16) except that yields were increased by growing overnight precultures at 25°C. The T domains were purified from the soluble cytoplasmic fraction as described (9,15). Proteins were subjected to immobilized nickel affinity, size exclusion, and ion exchange chromatography.…”

“…Fluorescence Spectroscopy in Solution-Fluorescence measurements were performed with an FP-750 spectrofluorimeter (Jasco, Tokyo, Japan) as described previously (9). Proteins were diluted at a concentration of 1 M in 5 mM sodium citrate buffer, 200 mM NaCl at the indicated pH, 2 h before measurements.…”

“…In the presence of membranes however, the T domain binds to the phospholipid bilayer between pH 7 and pH 6. This interaction involves the C terminus of the domain, whereas the N terminus is more exposed to the solvent (9). Between pH 6 and pH 4, the protein rearranges its structure and penetrates deeply inside the membrane (8 -10).…”

The translocation domain of botulinum neurotoxin A moderates the propensity of the catalytic domain to interact with membranes at acidic pH

“…The emission spectra were recorded from 310 to 370 nm at a scan rate of 125 nm/min. The fluorescence intensity ratio at 360 nm over 320 nm (rFI 360/320 ) represents the average of three values obtained from emission spectra that were corrected for blank measurements [23,28,29].…”