The diphtheria toxin channel-forming T-domain translocates its own NH2-terminal region and the catalytic domain across planar phospholipid bilayers

doi:10.1016/S1438-4221(00)80059-4

International Journal of Medical Microbiology

Volume 290, Issues 4–5, October 2000, Pages 435-440

https://doi.org/10.1016/S1438-4221(00)80059-4 Get rights and content

Abstract

The T-domain of diphtheria toxin, which extends from residue 202 to 378, causes the translocation of the catalytic A fragment (residues 1–201) across endosomal membranes and also forms ion-conducting channels in planar phospholipid bilayers. The carboxy-terminal 57-amino acid segment (residues 322–378) in the T-domain is all that is required to form these channels, but its ability to do so is greatly augmented by the portion of the T-domain upstream from this. Here we show that in association with channel formation by the T-domain, its hydrophilic 63-amino acid NH₂-terminal region (residues 202–264) as well as the entire catalytic A fragment (residues 1–201) cross the lipid bilayer. The phenomenon that enabled us to demonstrate this was the rapid closure of channels at cis negative voltages when a histidine tag was placed at various positions in the NH₂-terminal region of the T-domain or in the A fragment; the inhibition of this effect by trans nickel established that the histidine tag was present on the trans side of the membrane. Thus, all of the machinery necessary to translocate the A fragment across membranes is built into the 114 residues at the carboxy-terminal end of the T-domain (residues 265–378), without the requirement of any proteins in the plasma membrane (e. g., toxin receptor) or of any other cellular components.

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Cited by (16)

On the translocation of botulinum and tetanus neurotoxins across the membrane of acidic intracellular compartments
2016, Biochimica et Biophysica Acta - Biomembranes
Citation Excerpt :
Additional similarities between BoNT and SecY are their high content of α-helices and the fact that the SecY channel may open laterally to the lipid bilayer to release membrane spanning segment of a nascent protein. Also, the diphtheria toxin translocating domain is mainly α-helical and there is evidence that a single molecule is involved in the process with few transmembrane helices forming an ion channel [70,71]. At variance, it was recently reported that a BoNT/B trimer forms the protein conducting transmembrane channel in PC12 cells [35].
Tetanus and botulinum neurotoxins are produced by anaerobic bacteria of the genus Clostridium and are the most poisonous toxins known, with 50% mouse lethal dose comprised within the range of 0.1–few nanograms per Kg, depending on the individual toxin. Botulinum neurotoxins are similarly toxic to humans and can therefore be considered for potential use in bioterrorism. At the same time, their neurospecificity and reversibility of action make them excellent therapeutics for a growing and heterogeneous number of human diseases that are characterized by a hyperactivity of peripheral nerve terminals. The complete crystallographic structure is available for some botulinum toxins, and reveals that they consist of four domains functionally related to the four steps of their mechanism of neuron intoxication: 1) binding to specific receptors of the presynaptic membrane; 2) internalization via endocytic vesicles; 3) translocation across the membrane of endocytic vesicles into the neuronal cytosol; 4) catalytic activity of the enzymatic moiety directed towards the SNARE proteins.
Despite the many advances in understanding the structure–mechanism relationship of tetanus and botulinum neurotoxins, the molecular events involved in the translocation step have been only partially elucidated. Here we will review recent advances that have provided relevant insights on the process and discuss possible models that can be experimentally tested. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.
Deciphering Membrane Insertion of the Diphtheria Toxin T Domain by Specular Neutron Reflectometry and Solid-State NMR Spectroscopy
2009, Journal of Molecular Biology
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.
Bacterial Toxins Escape the Endosome by Inducing Vesicle Budding and Collapse
2021, ACS Chemical Biology
Botulinum neurotoxins: Biology, pharmacology, and toxicology
2017, Pharmacological Reviews
Cysteine modification: Probing channel structure, function and conformational change
2015, Advances in Experimental Medicine and Biology
Obstructing toxin pathways by targeted pore blockage
2012, Chemical Reviews

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The diphtheria toxin channel-forming T-domain translocates its own NH2-terminal region and the catalytic domain across planar phospholipid bilayers

Abstract

Refined structure of monomeric diphtheria toxin at 2.3 Å resolution

Protein Sci.

Membrane translocation and channel-forming activities of diphtheria toxin are blocked by replacing isoleucine 364 with lysine

Infect. Immun.

The entry of diphtheria toxin into the mammalian cell cytoplasm: evidence for lysosomal involvement

J. Cell. Biol.

Diphtheria toxin at low pH depolarizes the membrane, increases the membrane conductance and induces a new type of ion channel in Vero cells

EMBO J.

Replacement of negative by positive charges in the presumed membrane-inserted part of diphtheria toxin B fragment: Effect on membrane translocation and on formation of channels

J. Biol. Chem.

Probing the structure of the diphtheria toxin channel: Reactivity in planar lipid bilayer membranes of cysteine-substituted mutant channels with methane thiosulfonate derivatives

J. Gen. Physiol.

Diphtheria toxin fragment forms large pores in phospholipid bilayer membranes

Proc. Natl. Acad. Sci. USA

Structure-function relationship of the ion channel formed by diphtheria toxin in Vero cell membranes

J. Membr. Biol.

The diphtheria toxin channel-forming T-domain translocates its own NH₂-terminal region and the catalytic domain across planar phospholipid bilayers