Diphtheria toxin: nucleotide binding and toxin heterogeneity.

Area of Science:

• Biochemistry
• Molecular Biology
• Toxicology

Background:

• Diphtheria toxin is a potent bacterial toxin responsible for diphtheria.
• Understanding the molecular mechanisms of toxin-cell interaction is crucial for developing effective antitoxins and therapies.

Purpose of the Study:

• To investigate the binding of ATP and related compounds to diphtheria toxin.
• To identify and characterize new binding sites on the toxin molecule.
• To elucidate the role of these sites in toxin-cell interactions.

Main Methods:

• Flow dialysis was employed to study the binding kinetics of ATP and related nucleotides to diphtheria toxin.
• ATP-Sepharose chromatography was used to assess nucleotide binding and affinity.

Main Results:

• A novel ATP-binding site, termed the P site, was identified on diphtheria toxin, distinct from the NAD+-binding site on fragment A.
• The affinity of nucleotides for the P site correlated with their ability to inhibit toxin attachment to cell surfaces and cellular toxicity.
• A significant fraction of purified diphtheria toxin preparations contained an endogenous, tightly bound nucleotide, contributing to observed heterogeneity.

Conclusions:

• The P site on diphtheria toxin likely represents the binding site for cell surface receptors, mediating toxin-cell interactions.
• Nucleotide binding affinity is influenced by the number of phosphate groups and both nucleoside and phosphate components.
• The presence of endogenous nucleotides in toxin preparations can explain variations in physical and biological properties.