Galectin-1, a member of the conserved family of carbohydrate-binding proteins with affinity for β-galactosides, is a key modulator of diverse cell functions such as immune response and regulation. The binding affinity and specificity of galectin-1 for eight different β-galactosyl terminal disaccharides was studied using molecular-dynamics simulations in which the ligand was pulled away from the binding site using a mechanical force. We present what we believe to be a novel procedure, based on combinations of multistep trajectories, that was used to estimate the binding free energy (ΔG) of each disaccharide. The computed binding free energy differences show excellent correlation with experimental values determined previously. The small differences in affinity among the disaccharides are the result of an exquisite balance between the strengths of the galectin-sugar H-bonds and the H-bonds the protein and the disaccharides make with the solvent. Analysis of the free energies along the reaction coordinate shows that disaccharide unbinding/binding presents no energetic barrier and, therefore, is diffusion-limited. In addition, the calculations revealed that as the ligand is undocked from the binding site, breaking of protein-disaccharide H-bonds takes place in stages with intermediate states in which the interactions are bridged by water molecules.
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