Galactose-binding proteins characterize an important subgroup of sugar-binding proteins that are involved in a variety of biological processes. Structural studies have shown that the Gal-specific proteins encompass a diverse range of primary and tertiary structures. The binding sites for galactose also seem to vary in different protein-galactose complexes. No common binding site features that are shared by the Gal-specific proteins to achieve ligand specificity are so far known. With the assumption that common recognition principles will exist for common substrate recognition, the present study was undertaken to identify and characterize any unique galactose-binding site signature by analyzing the three-dimensional (3D) structures of 18 protein-galactose complexes. These proteins belong to 7 nonhomologous families; thus, there is no sequence or structural similarity across the families. Within each family, the binding site residues and their relative distances were well conserved, but there were no similarities across families. A novel, yet simple, approach was adopted to characterize the binding site residues by representing their relative spatial dispositions in polar coordinates. A combination of the deduced geometrical features with the structural characteristics, such as solvent accessibility and secondary structure type, furnished a potential galactose-binding site signature. The signature was evaluated by incorporation into the program COTRAN to search for potential galactose-binding sites in proteins that share the same fold as the known galactose-binding proteins. COTRAN is able to detect galactose-binding sites with a very high specificity and sensitivity. The deduced galactose-binding site signature is strongly validated and can be used to search for galactose-binding sites in proteins. PROSITE-type signature sequences have also been inferred for galectin and C-type animal lectin-like fold families of Gal-binding proteins.
The presence of an aromatic residue (Trp, Phe, Tyr) facing the nonpolar face of galactose is a common feature of galactose-specific lectins. The interactions such as those between the C-H groups of galactose and the pi-electron cloud of aromatic residues have been characterized as weak hydrogen bonds between soft acids and soft bases, largely governed by dispersive and charge transfer interactions. An analysis of the binding sites of several galactose-specific lectins revealed that the spatial position-orientation of galactose relative to the binding site aromatic residue varies substantially. The effect of variations in position-orientations of galactose on the interaction energies of galactose-aromatic residue complexes has not been determined so far. In view of this, MP2/6-311G++** calculations were performed on galactose- and glucose-aromatic residue analogue complexes in eight position-orientations. The results show that the strength of the C-H...pi interactions in galactose-aromatic residue complexes is comparable to that of a hydrogen bond. Rather than the type of aromatic residue, the position-orientation of the saccharide appears to be more critical in determining the strength of their interactions. Earlier studies have found the binding site aromatic residue to be critical, but its role was not clear. This study shows that the aromatic residue is important for discriminating galactose from glucose, in addition to its contribution to binding energy.
An aromatic amino acid is present in the binding site of a number of sugar binding proteins. The interaction of the saccharide with the aromatic residue is determined by their relative position as well as orientation. The position-orientation of the saccharide relative to the aromatic residue was found to vary in different sugar-binding proteins. In the present study, interaction energies of the complexes of galactose (Gal) and of glucose (Glc) with aromatic residue analogs have been calculated by ab initio density functional (U-B3LYP/ 6-31G**) theory. The position-orientations of the saccharide with respect to the aromatic residue observed in various Gal-, Glc-, and mannose-protein complexes were chosen for the interaction energy calculations. The results of these calculations show that galactose can interact with the aromatic residue with similar interaction energies in a number of position-orientations. The interaction energy of Gal-aromatic residue analog complex in position-orientations observed for the bound saccharide in Glc/Man-protein complexes is comparable to the Glc-aromatic residue analog complex in the same position-orientation. In contrast, there is a large variation in interaction energies of complexes of Glc-and of Gal-with the aromatic residue analog in position-orientations observed in Gal-protein complexes. Furthermore, the conformation wherein the O6 atom is away from the aromatic residue is preferred for the exocyclic -CH 2 OH group in Galaromatic residue analog complexes. The implications of these results for saccharide binding in Gal-specific proteins and the possible role of the aromatic amino acid to ensure proper positioning and orientation of galactose in the binding site have been discussed.
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