Galectins are a family of
lectins with a conserved
carbohydrate recognition domain that interacts with beta-
galactosides. By binding cell surface
glycoconjugates,
galectin-1 (gal-1) is involved in cell adhesion and migration processes and is an important regulator of
tumor angiogenesis. Here, we used heteronuclear NMR spectroscopy and molecular modeling to investigate
lactose binding to
gal-1 and to derive
solution NMR structures of
gal-1 in the
lactose-bound and unbound states. Structure analysis shows that the beta-strands and loops around the
lactose binding site, which are more open and dynamic in the unbound state, fold in around the bound
lactose molecule, dampening internal motions at that site and increasing motions elsewhere throughout the
protein to contribute entropically to the binding free energy. CD data support the view of an overall more open structure in the
lactose-bound state. Analysis of heteronuclear single quantum coherence titration binding data indicates that
lactose binds the two
carbohydrate recognition domains of the
gal-1 dimer with negative cooperativity, in that the first
lactose molecule binds more strongly (K(1)=21+/-6 x 10(3) M(-1)) than the second (K(2)=4+/-2 x 10(3) M(-1)). Isothermal calorimetry data fit using a sequential binding model present a similar picture, yielding K(1)=20+/-10 x 10(3) M(-1) and K(2)=1.67+/-0.07 x 10(3) M(-1). Molecular dynamics simulations provide insight into structural dynamics of the half-loaded
lactose state and, together with NMR data, suggest that
lactose binding at one site transmits a signal through the beta-sandwich and loops to the second binding site. Overall, our results provide new insight into
gal-1 structure-function relationships and to
protein-
carbohydrate interactions in general.