title: Membrane Interaction of botulinum neurotoxin A translocation (T) domain. The belt region is a regulatory loop for membrane interaction.
abstract: The translocation of the catalytic domain through the membrane of the endosome to the cell cytoplasm is a key step of intoxication by botulinum neurotoxin (BoNT). This step is mediated by the translocation (T) domain upon endosome acidification, although the mechanism of interaction of the T domain with the membrane is still poorly understood. Using physicochemical approaches and spectroscopic methods, we studied the interaction of the BoNT/A T domain with the membrane as a function of pH. We found that the interaction with membranes does not involve major secondary or tertiary structural changes, as reported for other toxins like diphtheria toxin. The T domain becomes insoluble around its pI value and then penetrates into the membrane. At that stage, the T domain becomes able to permeabilize lipid vesicles. This occurs for pH values lower than 5.5, in agreement with the pH encountered by the toxin within endosomes. Electrostatic interactions are also important for the process. The role of the so-called belt region was investigated with four variant proteins presenting different lengths of the N-extremity of the T domain. We observed that this part of the T domain, which contains numerous negatively charged residues, limits the protein-membrane interaction. Indeed, interaction with the membrane of the protein deleted of this extremity takes place for higher pH values than for the entire T domain. Overall, the data suggest that acidification eliminates repulsive electrostatic interactions between the T domain and the membrane, allowing its penetration into the membrane without triggering detectable structural changes.
authors.affiliation: Commissariat à l'Energie Atomique (CEA), Institut de Biologie et Technologies de Saclay (iBiTecS), Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), F-91191 Gif sur Yvette, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Galloux
authors.affiliation: Institut de Recherche en Technologies et Sciences pour le Vivant (IRTSV), Laboratoire de Chimie Biologie des Métaux (LCBM), UMR CEA-CNRS-UJF 5249, F-38054 Grenoble, France.
authors.forename: Heidi
authors.initials: H
authors.lastname: Vitrac
authors.affiliation: Institut de Recherche en Technologies et Sciences pour le Vivant (IRTSV), Laboratoire de Chimie Biologie des Métaux (LCBM), UMR CEA-CNRS-UJF 5249, F-38054 Grenoble, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Montagner
authors.affiliation: Commissariat à l'Energie Atomique (CEA), Institut de Biologie et Technologies de Saclay (iBiTecS), Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), F-91191 Gif sur Yvette, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Raffestin
authors.affiliation: Institut Pasteur, Unité des Bactéries Anaérobies et Toxines, 75724 Paris cedex 15, France.
authors.forename: Michel R
authors.initials: MR
authors.lastname: Popoff
authors.affiliation: Unité de Biochimie des Interactions Moléculaires, URA CNRS 2185, Département de Biologie Structurale et Chimie, 25-28 rue du Dr Roux, 75724 Paris cedex 15, France.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Chenal
authors.affiliation: Institut de Recherche en Technologies et Sciences pour le Vivant (IRTSV), Laboratoire de Chimie Biologie des Métaux (LCBM), UMR CEA-CNRS-UJF 5249, F-38054 Grenoble, France. Electronic address: vincent.forge@cea.fr.
authors.forename: Vincent
authors.initials: V
authors.lastname: Forge
authors.affiliation: Commissariat à l'Energie Atomique (CEA), Institut de Biologie et Technologies de Saclay (iBiTecS), Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), F-91191 Gif sur Yvette, France. Electronic address: daniel.gillet@cea.fr.
authors.forename: Daniel
authors.initials: D
authors.lastname: Gillet
mesh_terms: D019274:Botulinum Toxins, Type A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008567:Membranes, Artificial; D011485:Protein Binding; D020836:Protein Structure, Quaternary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity
chemical_list: D008567:Membranes, Artificial; D011994:Recombinant Proteins; D019274:Botulinum Toxins, Type A