IMMUNOCHEMICAL ANALYSIS OF THE STRUCTURE OF DIPHTHERIA-TOXIN SHOWS ALL 3 DOMAINS UNDERGO STRUCTURAL-CHANGES AT LOW PH

Diphtheria toxin is a bacterial protein that undergoes a physiological ly critical conformational change at low pH, This change involves a pa rtial unfolding event forming a molten globule-like structure, which e xposes hydrophobic regions and which allows the toxin to insert into, and translocate across, membranes, In this report, antibody binding wa s used to examine the regions of the toxin that undergo structural cha nges at low pH. Monoclonal antibodies specific to the catalytic (C), t ransmembrane (T), and receptor-binding (R) domains of diphtheria toxin were prepared and isolated, In addition, the binding of anti-peptide antibodies raised against pep tides in the C and T domains to toxin wa s examined, Anti-C monoclonals and antipeptide antibodies were found t o bind preferentially to low pH treated toxin relative to native toxin , Anti T and anti-R monoclonal binding ranged between preference for n ative toxin and preference for low pH-treated toxin, These results sug gest 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 conformati onal changes at low pH, Based on these results, a model in which low p H induces several coordinated changes in intra- and inter-domain inter actions is suggested, The participation of the R domain in these chang es is of particular significance because it suggests that the R domain plays a more important role in low pi-I-induced changes than previous ly realized.