A calorimetric study of the relationships between structure and function in diphtheria toxin

The pH and temperature stability of diphtheria toxin and its fragments have been studied by high sensitivity differential scanning calorimetry. These studies demonstrate that the pH-induced conformational transition associated with the mechanism of membrane insertion and translocation of the toxin involves a massive unfolding of the toxin molecule. At physiological temperatures (37°C), this process is centered at pH 4.7 at low ionic strength and at pH 5.4 in the presence of 0.2 M NaCl. At pH 8, the thermal unfolding of the nucleotide-bound toxin is centered at 58.2°C whereas that of the nucleotide-free toxin is centered at 51.8°C, indicating that nucleotide binding (ApUp) stabilizes the native conformation of the toxin. The unfolding profile of the toxin is consistent with two transitions most likely corresponding to the A fragment (Tm = 54.5°C) and the B fragment (Tm = 58.4°C), as inferred from experiments using the isolated A fragment. These two transitions are not independent, judging from the fact that the isolated A fragment unfolds at much lower temperatures (Tm = 44.2°C) and that the B fragment is insoluble in aqueous solutions when separated from the A fragment. Interfragment association contributes an extra -2.6 kcal/mol to the free energy of stabilization of the A fragment. Whereas the unfolding of the entire toxin is irreversible, the unfolding of the A fragment is a reversible process. These findings provide a thermodynamic basis for the refolding of the A fragment after reexposure to neutral pH immediately following translocation across the lysosomal membrane. The use of guanidine hydrochloride (GuHCl) as a denaturant has allowed the structure of the toxin to be further resolved. GuHCl destabilizes the two domains differently, allowing the thermodynamic parameters associated with the two domains to be measured with greater accuracy than was possible with the pH studies. These studies have allowed us to develop a complete thermodynamic structural model of the toxin which accounts for its temperature and GuHCl behavior. These studies demonstrate the existence of an intermediate physical state in which the low enthalpy domain is in the unfolded state while the high enthalpy domain remains folded. Furthermore, denaturation models in which the energetics of the interdomain interactions are explicitly considered show little difference in comparison to models which do not consider these terms. The relevance of these observations to the translocation process of the A fragment into the cytosol are discussed.