The inhibition of protein−carbohydrate interaction provides a powerful therapeutic strategy for the treatment of myriad human diseases. To date, application of such approaches have been frustrated by the inherent low affinity of carbohydrate ligands for their protein receptors. Because lectins typically exist in multimeric assemblies, a variety of polyvalent saccharide ligands have been prepared in the search for high affinity. The cluster glycoside effect, or the observation of high affinity derived from multivalency in oligosaccharide ligands, apparently represents the best strategy for overcoming the “weak binding” problem. Here we report the synthesis of a series of multivalent dendritic saccharides and a biophysical evaluation of their interaction with the plant lectin concanavalin A. Although a 30-fold enhancement in affinity on a valence-corrected basis is observed by agglutination assay, calorimetric titration of soluble protein with a range of multivalent ligands reveals no enhancement in binding free energies. Rather, IC50 values from agglutination measurements correlate well with entropies of binding. This observation suggests that hemagglutination measures a phenomenon distinct from binding that is typified by a large favorable entropy and an unfavorable enthalpy:  this process is almost certainly aggregation. Supporting this assertion, we report crystal structures of multivalent ligands cross-linking concanavalin A dimers. To the best of our knowledge, these structures are the first reported of their kind. Our results indicate that hemagglutination assays evaluate the ability of ligands to inhibit the formation of cross-linked lattices, a process only tangentially related to reversible ligand binding. Cluster glycoside effects observed in agglutination assays must, therefore, be viewed with caution. Such effects may or may not be relevant to the design of therapeutically useful saccharides.

Carbohydrate recognition by proteins is a key event in many biological processes. Concanavalin A is known to specifically recognize the pentasaccharide core (beta-GlcNAc-(1-->2)-alpha- Man-(1-->3)-[beta-GlcNAc-(1-->2)-alpha-Man-(1-->6)]-Man) of N-linked oligosaccharides with a Ka of 1.41 x 10(6 )M-1. We have determined the structure of concanavalin A bound to beta-GlcNAc-(1-->2)-alpha-Man-(1-->3)-[beta-GlcNAc-(1-->2)-alpha-Man- (1-->6)]-Man to 2.7A. In six of eight subunits there is clear density for all five sugar residues and a well ordered binding site. The pentasaccharide adopts the same conformation in all eight subunits. The binding site is a continuous extended cleft on the surface of the protein. Van der Waals interactions and hydrogen bonds anchor the carbohydrate to the protein. Both GlcNAc residues contact the protein. The GlcNAc on the 1-->6 arm of the pentasaccharide makes particularly extensive contacts and including two hydrogen bonds. The binding site of the 1-->3 arm GlcNAc is much less extensive. Oligosaccharide recognition by Con A occurs through specific protein carbohydrate interactions and does not require recruitment of adventitious water molecules. The beta-GlcNAc-(1-->2)-Man glycosidic linkage PSI torsion angle on the 1-->6 arm is rotated by over 50 degrees from that observed in solution. This rotation is coupled to disruption of interactions at the monosaccharide site. We suggest destabilization of the monosaccharide site and the conformational strain reduces the free energy liberated by additional interactions at the 1-->6 arm GlcNAc site.

We have determined the crystal structure of the methyl glycoside of Man alpha1-2 Man in complex with the carbohydrate binding legume lectin concanavalin A (Con A). Man alpha1-2 Man alpha-OMe binds more tightly to concanavalin A than do its alpha1-3 and alpha1-6 linked counterparts. There has been much speculation as to why this is so, including a suggestion of the presence of multiple binding sites for the alpha1-2 linked disaccharide. Crystals of the Man alpha1-2 Man alpha-OMe-Con A complex form in the space group P2(1)2(1)2(1) with cell dimensions a = 119.7 A, b = 119.7 A, c = 68.9 A and diffract to 2. 75A. The final model has good geometry and an R factor of 19.6% (Rfree= 22.8%). One tetramer is present in the asymmetric unit. In three of the four subunits, electron density for the disaccharide is visible. In the fourth only a monosaccharide is seen. In one subunit the reducing terminal sugar is recognized by the monosaccharide site; the nonreducing terminal sugar occupies a new site and the major solution conformation of the inter-sugar glycosidic linkage conformation is adopted. In contrast, in another subunit the non reducing terminal sugar sits in the so called monosaccharide binding site; the reducing terminal sugar adopts a different conformation about its inter-sugar glycosidic linkage in order for the methyl group to access a hydrophobic pocket. In the third subunit, electron density for both binding modes is observed. We demonstrate that an extended carbohydrate binding site is capable of binding the disaccharide in two distinct ways. These results provide an insight in to the balance of forces controlling protein carbohydrate interactions.

Crystals have been obtained of succinylated concanavalin A complexed to a novel bidentate synthetic ligand. The crystals are the first example of a lectin with a synthetic multivalent ligand and the first report of crystallization of succinylated concanavalin A. The crystals were obtained by sitting-drop vapour diffusion equilibrating with a solution of 20% polyethylene glycol, pH 5,293.5 K. Crystals are orthorhombic, belonging to space group C222~ with unit-cell dimensions of a = 99.1, b = 127.4, c = 118.9 A. The asymmetric unit contains a dimer, with over 65% of the volume occupied by water. The ligand cross links concanavalin A monomers. Succinylated concanavalin A is known to be a dimcr in solution, yet it is found as the typical concanavalin A tetramer in the crystal. The contacts holding together the tetramer appear extensive and suggest that a fine balance between dimer and tetramers exists. Data to 2.65 A, have been collected and the structure determined by the molecular replacement method.