Molecular recognition of sialyl Lewis(x) and related saccharides by two lectins

The interaction of sialyl Lewis(x), Lewis(x), and alpha -L-Fuc-(1 -->3)-bet a -D-GlcNAc with isolectin A from Lotus tetragonolobus (LTL-A), and with Al euria aurantia agglutinin (AAA) was studied using NMR experiments and surfa ce plasmon resonance. Both lectins are specific for fucose residues. From N MR experiments it was concluded that alpha -L-Fuc-(1 -->3)-P-D-GlcNAc and L ewis(x) bound to both lectins, whereas sialyl Lewis(x) only bound to AAA. I ncreased line broadening of H-1 NMR signals of the carbohydrate ligands upo n binding to AAA and LTL-A suggested that AAA bound to the ligands more tig htly. Further comparison of line widths showed that for both lectins bindin g strengths decreased from alpha -L-Fuc-(1 -->3)-beta -D-GlcNAc to Lewis(x) and were lowest for sialyl Lewis(x). Surface plasmon resonance measurement s were then employed to yield accurate dissociation constants. TrNOESY, QUI ET-trNOESY, and trROESY experiments delivered bioactive conformations of th e carbohydrate ligands, and STD NMR experiments allowed a precise epitope m apping of the carbohydrates bound to the lectins. The bioactive conformatio n of Lewis(x) bound to LTL-A, or AAA revealed an unusual orientation of the fucose residue, with negative values for both dihedral angles, phi and psi , at the alpha (1 --> -3)-glycosidic linkage. A similar distortion of the f ucose orientation was also observed for sialyl Lewis(x) bound to AAA. From STD NMR experiments it followed that only the L-fucose residues are in inti mate contact with the protein. Presumably steric interactions are responsib le for locking the sialic acid residue of sialyl Lewis(x) in one out of man y orientations that are present in aqueous solution. The sialic acid residu e of sialyl Lewis(x) bound to AAA adopts an orientation similar to that in the corresponding sialyl Lewis(x)/E-selectin complex.