Thermodynamics of Intersubunit Interactions in Cholera Toxin upon Binding to the Oligosaccharide Portion of Its Cell Surface Receptor, Ganglioside G_M1

The binding and the energetics of the interaction of cholera toxin with the oligosaccharide portion of ganglioside G_M1 (oligo-G_M1), the toxin cell surface receptor, have been studied by high-sensitivity isothermal titration calorimetry and differential scanning calorimetry. Previously, we have shown that the association of cholera toxin to ganglioside G_M1 enhances the cooperative interactions between subunits in the B-subunit pentamer [Goins, B., & Freire, E. (1988) Biochemistry 27, 2046-2052], New experiments presented in this paper reveal that the oligosaccharide portion of the receptor is by itself able to enhance the intersubunit cooperative interactions within the B pentamer. This effect is seen in the protein unfolding transition as a shift from independent unfolding of the B promoters toward a cooperative unfolding. To identify the origin of this effect, the binding of cholera toxin to oligo-G_M1 has been measured calorimetrically under isothermal conditions. The binding curve at 37 °C is sigmoidal, indicating cooperative binding. The binding data can be described in terms of a nearest-neighbor cooperative interaction binding model. In terms of this model, the association of a oligo-G_M1 molecule to a B protomer affects the association to adjacent B promoters within the pentameric ring. The measured intrinsic binding enthalpy per protomer is -22 kcal/mol and the cooperative interaction enthalpy -11 kcal/mol. The intrinsic binding constant determined calorimetrically is 1.05 × 106 M^-1 at 37 °C and the cooperative Gibbs free energy equal to -850 cal/mol. These studies provide the first direct thermodynamic description of the cooperative binding of a protein to its cell surface receptor and have allowed us to place energetics constraints on the putative changes in protein conformation triggered by the intrinsic receptor binding process.