title: Deciphering membrane insertion of the diphtheria toxin T domain by specular neutron reflectometry and solid-state NMR spectroscopy.
abstract: Insertion and translocation of soluble proteins into and across biological membranes are involved in many physiological and pathological processes, but remain poorly understood. Here, we describe the pH-dependent membrane insertion of the diphtheria toxin T domain in lipid bilayers by specular neutron reflectometry and solid-state NMR spectroscopy. We gained unprecedented structural resolution using contrast-variation techniques that allow us to propose a sequential model of the membrane-insertion process at angstrom resolution along the perpendicular axis of the membrane. At pH 6, the native tertiary structure of the T domain unfolds, allowing its binding to the membrane. The membrane-bound state is characterized by a localization of the C-terminal hydrophobic helices within the outer third of the cis fatty acyl-chain region, and these helices are oriented predominantly parallel to the plane of the membrane. In contrast, the amphiphilic N-terminal helices remain in the buffer, above the polar headgroups due to repulsive electrostatic interactions. At pH 4, repulsive interactions vanish; the N-terminal helices penetrate the headgroup region and are oriented parallel to the plane of the membrane. The C-terminal helices penetrate deeper into the bilayer and occupy about two thirds of the acyl-chain region. These helices do not adopt a transmembrane orientation. Interestingly, the T domain induces disorder in the surrounding phospholipids and creates a continuum of water molecules spanning the membrane. We propose that this local destabilization permeabilizes the lipid bilayer and facilitates the translocation of the catalytic domain across the membrane.
authors: Institut Pasteur, CNRS URA, Département de Biologie Structurale et Chimie, Paris, France.
authors: Alexandre
authors: A
authors: Chenal
authors: Lydia
authors: L
authors: Prongidi-Fix
authors: Aurélie
authors: A
authors: Perier
authors: Christopher
authors: C
authors: Aisenbrey
authors: Grégory
authors: G
authors: Vernier
authors: Stephan
authors: S
authors: Lambotte
authors: Michael
authors: M
authors: Haertlein
authors: Marie-Thérèse
authors: MT
authors: Dauvergne
authors: Giovanna
authors: G
authors: Fragneto
authors: Burkhard
authors: B
authors: Bechinger
authors: Daniel
authors: D
authors: Gillet
authors: Vincent
authors: V
authors: Forge
authors: Michel
authors: M
authors: Ferrand
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D004167:Diphtheria Toxin / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009502:Neutrons; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary
chemical_list: D004167:Diphtheria Toxin; D008051:Lipid Bilayers