We report here solution NMR relaxation measurements that show millisecond time-scale intersubunit dynamics in the homopentameric B subunit (VTB) of the toxin derived from Escherichia coli O157. These data are consistent with interconversion between an axially symmetric form and a low-abundance (similar to10%, 45 degreesC) higher energy form. The higher energy state is depopulated on binding of a novel bivalent analogue (P-k dimer) of the natural carbohydrate acceptor globotriaosylceramide. The isothermal titration calorimetry isotherm for the binding of P-k dimer to VTB is consistent with a five-site sequential binding model which assumes that cooperative effects arise through communication only between neighboring binding sites. The resulting thermodynamic parameters (K-a1 = 114 +/- 2.2 M-1, K-a2 = 283 +/- 4.5 M-1, DeltaH(1)degrees = -116.3 +/- 0.55 kJ/mol, and DeltaH(2)degrees = -50.3 +/- 0.11 kJ/mol) indicate favorable entropic cooperativity that has not previously been observed in multivalent systems.