Fig. 1. Distant homologs of diphtheria toxin retain the domain architecture of DT.

a DT intoxication pathway. Briefly, the C-terminal Receptor binding domain of DT (blue) triggers receptor mediated endocytosis upon binding its receptor Heparin Binding EGF Like Growth Factor (HBEGF). In the endosome, a furin-like protease cleaves between the Cytotoxic domain (red) and the Translocation domain (yellow) which remain tethered by an intramolecular disulfide bond. Acidification due to endosomal maturation results in conformational changes in the T domain which then inserts into the membrane, facilitating the escape of the C domain into the cytosol. The reducing environment of the cytosol reduces the disulfide bond, freeing the C domain to ADP-ribosylate its target eEF-2. ADP-ribosylation of the diphthamide residue on eEF-2 inactivates it, leading to protein synthesis inhibition and cell death. b Phylogenetic tree. DT and DT-like proteins from S. albireticuli (DT-like S.a) and S. peptonophila (DT-like S.p) are highlighted in gray, green and blue, respectively. Clade support values correspond to maximum likelihood bootstrap values from RAxML and posterior probability percentages from MrBayes. c Domain architecture and residue boundaries of DT, S.a, and S.p. d Three-way sequence identity and similarity (brackets) between DT, S.a, and S.p. e Purity of DT and DT-like proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis. f Melting temperature of DT, S.a and S.p at pH 7.5 as determined by differential scanning fluorimetry (mean ± SD, n = 4); n = 3 biological replicates. g pH stability of DT, S.a and S.p across a range of pHs measured using differential scanning fluorimetry (mean ± SD, n = 4); n = 3 biological replicates.