Role of water in the specific binding of mannose and mannooligosaccharides to concanavalin A

We report here, the first solution state evidence for the role of water molecules in the specific interaction of carbohydrates with a legume lectin, concanavalin A. Concanavalin A from Canavalia ensiformis is a protein containing 237 amino acid residues with each monomer possessing one sugar binding site as well as sites for transition-metal ions, Mn2+ and Ca2+. The lectin binds specifically to alpha-anomers of monosaccharides, D-glucopyranoside and D-mannopyranoside, and recognizes the trimannosidic core of N-linked glycoproteins, 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside with high specificity, which constitutes the minimum carbohydrate epitope that completely fills the sugar binding site. Sensitive isothermal titration microcalorimetry coupled with osmotic stress strategy on concanavalin A was used to dissect out the differential involvement of water molecules in the recognition of the branched trimannoside (3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside), the individual dimannosidic arms (3-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside and 6-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside) as well as the monomer unit, D-mannopyranoside, The specific binding of concanavalin A to different sugars, is accompanied by differential uptake of water molecules during the binding process. These results not only complement the X-ray crystallographic studies of legume lectin-sugar complexes displaying structurally conserved water molecules mediating the specific ligation of the sugars with the corresponding sites in the binding pocket but also provide a rationale for the observed compensatory behavior of enthalpies with entropies in lectin-sugar interactions.