Protein-bile acid interactions are crucial microscopic events at the basis of both physiological and pathological biochemical pathways. Bile acid binding proteins are intracellular transporters able to bind ligands with different stoichiometry, selectivity, and cooperativity. The molecular determinants and energetics of interaction are the observables that connect the microscopic to the macroscopic frameworks. This paper addresses the study and proposes a mechanism for the multi-site interaction of bile acids with chicken ileal bile acid binding protein (I-BABP) with the aim of elucidating the determinants of ligand binding in comparison to homologous proteins from different species and tissues. A thermodynamic binding model describing two independent consecutive binding sites is derived from isothermal titration calorimetry experiments and validated on the basis of both protein-observed and ligand-observed NMR titration data. It emerges that a singly bound protein is relatively abundant at low ligand:protein molar ratios assessing the absence of strong cooperativity. Both the measured energetics of binding and the distributed protein chemical shift perturbations are in agreement with a first binding event triggering a global structural rearrangement. The enthalpic and entropic contributions associated with binding of the first ligand indicate that the interaction increases stability and order of the bound protein. The described results point to the presence of a protein scaffold which is able to establish long-range communication networks, but does not manifest positive binding cooperativity, as observed for the human protein. We consider chicken I-BABP a suitable model to address the molecular basis for a gain of function on going from non-mammalian to mammalian species.
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