No abstract

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.

The antigenic recognition of Shigella flexneri O-polysaccharide, which consists of a repeating unit ABCD [-->2)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-beta-D-GlcpNAc-(1-->], by the monoclonal antibody SYA/J6 (IgG3, kappa) has been investigated by crystallographic analysis of the Fab domain and its two complexes with two antigen segments (a pentasaccharide Rha A-Rha B-Rha C-GlcNAc D-Rha A' and a modified trisaccharide Rha B-Rha C-GlcNAc D in which Rha C* is missing a C2-OH group). These complex structures, the first for a Fab specific for a periodic linear heteropolysaccharide, reveal a binding site groove (between the V(H) and V(L) domains) that makes polar and nonpolar contacts with all the sugar residues of the pentasaccharide. Both main-chain and side-chain atoms of the Fab are used in ligand binding. The charged side chain of Glu H50 of CDR H2 forms crucial hydrogen bonds to GlcNAc of the oligosaccharides. The modified trisaccharide is more buried and fits more snugly than the pentasaccharide. It also makes as many contacts (approximately 75) with the Fab as the pentasaccharide, including the same number of hydrogen bonds (eight, with four being identical). It is further engaged in more hydrophobic interactions than the pentasaccharide. These three features favorable to trisaccharide binding are consistent with the observation of a tighter complex with the trisaccharide than the pentasaccharide. Thermodynamic data demonstrate that the native tri- to pentasaccharides have free energies of binding in the range of 6.8-7.4 kcal mol(-1), and all but one of the hydrogen bonds to individual hydroxyl groups provide no more than approximately 0.7 kcal mol(-1). They further indicate that hydrophobic interactions make significant contributions to binding and, as the native epitope becomes larger across the tri-, tetra-, pentasaccharide series, entropy contributions to the free energy become dominant.