Dissecting the Cholera Toxin−Ganglioside GM1 Interaction by Isothermal Titration Calorimetry

• Published in: Journal of the American Chemical Society Vol. 126; no. 4; pp. 1047 - 1054
• Main Authors: Turnbull, W. Bruce, Precious, Bernie L, Homans, Steve W
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 04.02.2004
• Subjects: Biological and medical sciences; Calorimetry - methods; Carbohydrate Sequence; Cholera Toxin - chemistry; Cholera Toxin - metabolism; Fluorescence; Fundamental and applied biological sciences. Psychology; G(M1) Ganglioside - chemistry; G(M1) Ganglioside - metabolism; Interactions. Associations; Intermolecular phenomena; Models, Molecular; Molecular biophysics; Molecular Sequence Data; Thermodynamics; Titrimetry; GM1 ganglioside; Toxin; Binding capacity; Chemical affinity; Thermodynamic parameter; Pentasaccharide; Molecular interaction; Thermochemical titration
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja0378207

The complex of cholera toxin and ganglioside GM1 is one of the highest affinity protein−carbohydrate interactions known. Herein, the GM1 pentasaccharide is dissected into smaller fragments to determine the contribution of each of the key monosaccharide residues to the overall binding affinity. Displacement isothermal titration calorimetry (ITC) has allowed the measurement of all of the key thermodynamic parameters for even the lowest affinity fragment ligands. Analysis of the standard free energy changes using Jencks' concept of intrinsic free energies reveals that the terminal galactose and sialic acid residues contribute 54% and 44% of the intrinsic binding energy, respectively, despite the latter ligand having little appreciable affinity for the toxin. This analysis also provides an estimate of 25.8 kJ mol-1 for the loss of independent translational and rotational degrees of freedom on complexation and presents evidence for an alternative binding mode for ganglioside GM2. The high affinity and selectivity of the GM1−cholera toxin interaction originates principally from the conformational preorganization of the branched pentasaccharide rather than through the effect of cooperativity, which is also reinvestigated by ITC.