Analysis of the binding specificities of oligomannoside-binding proteins using methylated monosaccharides.

The binding specificities of the closely related lectins from Canavalia ensiformis and Dioclea grandiflora were examined using specifically O-alkylated mono- and disaccharides. Both lectins accept any substitution at the monosaccharide C2 hydroxyl group. The binding energy of C2-alkylated ligands-concanavalin A complexes increases by 1 kcal mol-1 for the C2-O-ethyl ligand, while the binding energies of the corresponding complexes with the Dioclea lectin are identical. Both lectins accept methyl, but not ethyl, substitution of the C3 hydroxyl, in contrast to earlier reports. The results are interpreted in terms of existing models of the concanavalin A binding site. While the results are consistent with a model of the concanavalin A extended binding site that places the non-reducing terminus of all disaccharides in the monosaccharide binding site, they point to the dangers of interpreting the binding behavior of unnatural saccharide ligands on the basis of crystallographic data obtained with native ligands.

Calorimetric analysis of the binding of lectins with overlapping carbohydrate-binding ligand specificities.

The thermodynamics of binding of a system of plant lectins specific for the oligosaccharide methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside have been studied calorimetrically. This system of lectins consists of concanavalin A, the lectin isolated from Dioclea grandiflora, and the lectin from Galanthus nivalis. The group thus contains lectins with similar structures and similar binding properties as well as lectins with different structures but similar binding properties. Concanavalin A and the lectin from Dioclea are highly homologous, while the lectin from Galanthus nivalis shares no sequence homology with either of the legume lectins, although it also binds the mannose trisaccharide tightly. Calorimetric data for oligosaccharide binding to both of the legume lectins suggests that the total binding site comprises a single high-affinity site and an additional extended site. The pattern of binding for the lectin from Galanthus is significantly different. Binding studies with the same saccharides indicate that the lectin has binding sites designed specifically for the 1-->3 and 1-->6 arms of the mannose trisaccharide that are unable to accommodate other saccharides. Enthalpy--entropy compensation was observed for several saccharides as a function of lectin structure. Contributions of solvation effects to the enthalpy of binding and the configurational entropies were determined experimentally. For those systems studied here, solute-solute attractive interactions and configurational entropies were the greatest contributors to enthalpy-entropy compensation. Our studies clearly demonstrate that, despite their common affinity for the mannose trisaccharide, the three lectins bind oligosaccharides very differently.

Energetics of lectin-carbohydrate binding. A microcalorimetric investigation of concanavalin A-oligomannoside complexation.

Despite years of study, a comprehensive picture of the binding of the lectin from Canavalia ensiformis, concanavalin A, to carbohydrates remains elusive. We report here studies on the interaction of concanavalin A with methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, the minimum carbohydrate epitope that completely fills the oligosaccharide binding site, and the two conceptual disaccharide "halves" of the trisaccharide, methyl 3-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside and methyl 6-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, using titration microcalorimetry. In all cases the interaction of protein and carbohydrate is enthalpically driven, with an unfavorable entropic contribution. The choice of concentration scales has an important impact on both the magnitude and, in some cases, the sign of the entropic component of the free energy of binding. The thermodynamic data suggest binding of the two disaccharides may take place in distinct sites, as opposed to binding in a single high affinity site. In contrast to carbohydrate-antibody binding, delta Cp values were small and negative, pointing to possible differences in the motifs used by the two groups of proteins to bind carbohydrates. The thermodynamic data are interpreted in terms of solvent reorganization. Cooperativity during lectin-carbohydrate binding was also investigated. Significant cooperativity was observed only for binding of the trisaccharide, and gave a Hill plot coefficient of 1.3 for dimeric protein.