A conserved motif in transmembrane helix 1 of diphtheria toxin mediates catalytic domain delivery to the cytosol

Abstract

A 10-aa motif in transmembrane helix 1 of diphtheria toxin that is conserved in anthrax edema factor, anthrax lethal factor, and botulinum neurotoxin serotypes A, C, and D was identified by BLAST, CLUSTAL W, and MEME computational analysis. Using the diphtheria toxin-related fusion protein toxin DAB 389IL-2, we demonstrate that introduction of the L221E mutation into a highly conserved residue within this motif results in a nontoxic catalytic domain translocation deficient phenotype. To further probe the function of this motif in the process by which the catalytic domain is delivered from the lumen of early endosomes to the cytosol, we constructed a gene encoding a portion of diphtheria toxin transmembrane helix 1, T1, which carries the motif and is expressed from a CMV promoter. We then isolated stable transfectants of Hut102͞6TG cells that express the T1 peptide, Hut102͞6TG-T1. In contrast to the parental cell line, Hut102͞6TG-T1 cells are ca. 10 4 -fold more resistant to the fusion protein toxin. This resistance is completely reversed by coexpression of small interfering RNA directed against the gene encoding the T1 peptide in Hut102͞ 6TG-T1 cells. We further demonstrate by GST-DT140-271 pull-down experiments in the presence and absence of synthetic T1 peptides the specific binding of coatomer protein complex subunit ␤ to this region of the diphtheria toxin transmembrane domain.early endosomes ͉ translocation ͉ ADP-ribosyltransferase ͉ coatomer protein complex subunit ␤ T he intoxication of eukaryotic cells by diphtheria toxin follows an ordered series of interactions between the toxin and cellular factors that lead to inhibition of protein synthesis and cell death (1). Biochemical, genetic, and x-ray crystallographic analysis of the toxin has shown the protein to be composed of three distinct domains: an N-terminal catalytic (C) domain, a central transmembrane (T) domain, and the C-terminal receptor-binding domain (2-5). The intoxication process is initiated by the binding of the toxin to its cell surface receptor, a heparin binding epidermal growth factor-like precursor, and CD9 (6, 7). Once bound to its receptor, the toxin is internalized by receptormediated endocytosis into an early endosomal compartment (8). Upon acidification of the endosomal lumen by vesicular ATPase, the T domain undergoes a conformational change and spontaneously inserts into the vesicular membrane, forming an 18-to 22-Å pore or channel (9, 10). It is widely believed that the C domain, in a fully denatured form, is specifically thread through this channel and released into the cytosol. Once the C domain is refolded into an active conformation, it catalyzes the NAD ϩ -dependent ADP ribosylation of elongation factor 2 (EF-2), causing irreversible inhibition of protein synthesis and death of the cell by apoptosis (11,12). Although the endosomal membrane translocation of the C domain is understood in broad terms, the precise molecular mechanism(s) of this step in the intoxication process has remained largely unknown.Two hypotheses for C ...

Topography of the Hydrophilic Helices of Membrane-Inserted Diphtheria Toxin T Domain: TH1−TH3 as a Hydrophilic Tether

“…However, exactly how T domain assists translocation is unclear. Furthermore, translocation may be aided by cytoplasmic factors (25,26) Defining the structure of the membrane-inserted T domain is likely to provide insights into the mechanism of A chain translocation. The T domain is primarily comprised of 9 α-helices (TH1-9).…”

Increased RNAi is related to intracellular release of siRNA via a covalently attached signal peptide

“…The peptide TQIENLKEKG was identified by Ratts et al (2005) as a conserved 10 amino acid motif in several bacterial toxins, e.g., diphtheria toxin, anthrax edema factor, anthrax lethal factor, and botulinum toxins. This motif is part of a transmembrane helix within the toxin that facilitates translocation of the respective catalytic domains from transport vesicles into the cytoplasm where the toxin exerts its harmful effects (Falnes and Sandvig 2000).…”

Defensive strategies of Bacillus anthracis that promote a fatal disease

“…This charge-state Brownian ratchet mechanism would be reinforced by the refolding of translocated segments into structures too large to fit back into the channel [14]. This model is sufficient to explain the translocation process without the requirement of mammalian accessory factors, although it is possible that cytosolic factors facilitate the process in vivo, as has been observed for other toxins [16].…”