Deciphering membrane insertion of the diphtheria toxin T domain by specular neutron reflectometry and solid-state NMR spectroscopy.
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.
Chenal
Alexandre
A
Institut Pasteur, CNRS URA, Département de Biologie Structurale et Chimie, Paris, France.
Prongidi-Fix
Lydia
L
Perier
Aurélie
A
Aisenbrey
Christopher
C
Vernier
Grégory
G
Lambotte
Stephan
S
Haertlein
Michael
M
Dauvergne
Marie-Thérèse
MT
Fragneto
Giovanna
G
Bechinger
Burkhard
B
Gillet
Daniel
D
Forge
Vincent
V
Ferrand
Michel
M
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
D004167:Diphtheria Toxin; D008051:Lipid Bilayers