title: Accessibility changes within diphtheria toxin T domain upon membrane penetration probed by hydrogen exchange and mass spectrometry.
abstract: The translocation domain of diphtheria toxin inserts in membrane and becomes functional when the pH inside endosomes is acid. At that stage, the domain is in a partially folded state; this prevents the use of high-resolution methods for the characterization of its functional structure. On that purpose, we report here the use of hydrogen/deuterium exchange experiments coupled to mass spectrometry. The conformation changes during the different steps of insertion into lipid bilayer are monitored with a resolution of few residues. Three parts of the translocation domain can be distinguished. With a high protection against exchange, the C-terminal hydrophobic helical hairpin is embedded in the membrane. Despite a lower protection, a significant effect in the presence of lipid vesicles shows that the N-terminal part is in interaction with the membrane interface. The sensitivity to the ionic strength indicates that electrostatic interactions are important for the binding. The middle part of the domain has an intermediate protection; this suggests that this part of the domain can be embedded within the membrane but remains quite dynamic. These results provide unprecedented insight into the structure reorganization of the protein to go from a soluble state to a membrane-inserted one.
authors: Laboratoire de Spectrométrie de Masse des Protéines, Institut de Biologie Structurale, 41 rue Jules Horowitz, 38027 Grenoble, France.
authors: Petr
authors: P
authors: Man
authors: Caroline
authors: C
authors: Montagner
authors: Heidi
authors: H
authors: Vitrac
authors: Daniel
authors: D
authors: Kavan
authors: Sylvain
authors: S
authors: Pichard
authors: Daniel
authors: D
authors: Gillet
authors: Eric
authors: E
authors: Forest
authors: Vincent
authors: V
authors: Forge
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D041961:Deuterium Exchange Measurement; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D021381:Protein Transport; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D004167:Diphtheria Toxin; D008051:Lipid Bilayers; D006859:Hydrogen