1.C.7 The Diphtheria Toxin (DT) Family
The DT family consists of a single protein, diphtheria toxin (DT). DT is synthesized as a 535 amino acyl residue protein encoded by the tox gene of corynebacteriophage beta in Corynebacterium diphtheriae. The protein is secreted as a single polypeptide chain in a leader peptide-dependent process and is then cleaved between arg-193 and ser-194 by extracellular trypsin-like proteases. This cleavage event yields the N-terminal A chain that contains the NAD+:diphtheramide ADP ribosyl transferase (EC 18.104.22.168) activity, and the C-terminal B chain that forms the transmembrane channel that allows the A chain to enter the cytoplasm of the eukaryotic cell where the latter exerts its toxic effect by ADP ribosylating a histidyl residue on the N-terminal region of elongation factor 2 (EF2). In the extracellular environment, the A and B chains are held together by a disulfide bridge.
Diphtheria toxin serves as the prototype for two chain (or multidomain) bacterial and plant toxins. The B chain binds to the external plasma membrane receptor as a prelude to channel formation. The C-terminal half of the B chain is the receptor binding domain while the N-terminal half as well as part of the binding domain comprise the channel. While the translocation process and the structure of the membrane embedded channel are not understood in detail, translocation has been reported to require energy. Lai et al. (2008) reported that of the deeply inserted helices in the Diphtheria toxin T domain: helices 5, 8, and 9, interact strongly and promote pore formation, while helices 6/7 limit pore formation.
Other well-characterized toxins that have an AB structure, where B translocates A, include botulinum neurotoxins A-G, several chlostridial neurotoxins, two anthrax neurotoxins, and tetanus neurotoxin. Membrane insertion of the B chain and channel formation often occur after phagocytosis in vesicles or the endoplasmic reticulum where acid pH induces the conformational change that accompanies insertion into the endosomal membrane. After disulfide reduction, exposure to the neutral pH of the cytosol may trigger the final translocation of the A-chain. DT appears to form oligomers with variable stoichiometry and pore size. The greater the number of monomers, the larger the pore size (Sharpe and London, 1999).
The diphtheria toxin T domain helps translocate the A chain of the toxin across membranes. The membrane topography of the diphtheria toxin T domain linked to the A chain revealed a transient transmembrane hairpin and potential translocation mechanisms (Wang and London, 2009). Translocation of the diphtheria toxin catalytic domain from the lumen of early endosomes into the cytosol of eukaryotic cells requires the presence of coatomer protein complex I (COPI) via lysine residues in transmembrane helix 1 (Trujillo et al., 2010). The mechanism of diphtheria toxin delivery to the eukaryotic cell cytosol and the participatory cellular factors have been defined (Murphy, 2011).
The translocation domain (T-domain) of diphtheria toxin contains 10 α-helices in the aqueous crystal structure. Upon exposure to a planar lipid bilayer under acidic conditions, it inserts to form a channel and transports the attached amino-terminal catalytic domain across the membrane. Helices 5, 8, and 9 form transmembrane segments in the open-channel state, with helices 1-4 translocated across the membrane. Helices 6-7 also insert to form a constriction that occupies a small portion of the total channel length. Helix 5 may also have a transmembrane orientation and remains helical in the open-channel state; the middle of the helix may be aligned with the constriction in the channel (Kienker et al. 2015). The diphtheria toxin translocation domain has been studied dynamically and mutationally (Kyrychenko et al. 2018).
The transport reaction catalyzed by DT is:
A-chain (out) → A-chain (in)