Immunochemical analysis of the structure of diphtheria toxin shows all three domains undergo structural changes at low pH.
Diphtheria toxin is a bacterial protein that undergoes a physiologically critical conformational change at low pH. This change involves a partial unfolding event forming a molten globule-like structure, which exposes hydrophobic regions and which allows the toxin to insert into, and translocate across, membranes. In this report, antibody binding was used to examine the regions of the toxin that undergo structural changes at low pH. Monoclonal antibodies specific to the catalytic (C), transmembrane (T), and receptor-binding (R) domains of diphtheria toxin were prepared and isolated. In addition, the binding of anti-peptide antibodies raised against peptides in the C and T domains to toxin was examined. Anti-C monoclonals and antipeptide antibodies were found to bind preferentially to low pH-treated toxin relative to native toxin. Anti-T and anti-R monoclonal binding ranged between preference for native toxin and preference for low pH-treated toxin. These results suggest that the C domain becomes more exposed to solution at low pH, and that both the T and R domains of the B chain undergo major conformational changes at low pH. Based on these results, a model in which low pH induces several coordinated changes in intra- and inter-domain interactions is suggested. The participation of the R domain in these changes is of particular significance because it suggests that the R domain plays a more important role in low pH-induced changes than previously realized.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D006825:Hybridomas; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice / Q000276:immunology; D008961:Models, Structural; D010446:Peptide Fragments / Q000276:immunology; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D004167:Diphtheria Toxin; D000939:Epitopes; D010446:Peptide Fragments