Analysis of Glycan Recognition by Concanavalin A Using\nAbsolute Binding Free Energy Calculations

Carbohydrates are\nkey biological mediators of molecular recognition\nand signaling processes. In this case study, we explore the ability\nof absolute binding free energy (ABFE) calculations to predict the\naffinities of a set of five related carbohydrate ligands for the lectin\nprotein, concanavalin A, ranging from 27-atom monosaccharides to a\n120-atom complex-type N-linked glycan core pentasaccharide. ABFE calculations\nquantitatively rank and estimate the affinity of the ligands in relation\nto microcalorimetry, with a mean signed error in the binding free\nenergy of −0.63 ± 0.04 kcal/mol. Consequently, the diminished\nbinding efficiencies of the larger carbohydrate ligands are closely\nreproduced: the ligand efficiency values from isothermal titration\ncalorimetry for the glycan core pentasaccharide and its constituent\ntrisaccharide and monosaccharide compounds are respectively −0.14,\n−0.22, and −0.41 kcal/mol per heavy atom. ABFE calculations\npredict these ligand efficiencies to be −0.14 ± 0.02,\n−0.24 ± 0.03, and −0.46 ± 0.06 kcal/mol per\nheavy atom, respectively. Consequently, the ABFE method correctly\nidentifies the high affinity of the key anchoring mannose residue\nand the negligible contribution to binding of both β-GlcNAc\narms of the pentasaccharide. While challenges remain in sampling the\nconformation and interactions of these polar, flexible, and weakly\nbound ligands, we nevertheless find that the ABFE method performs\nwell for this lectin system. The approach shows promise as a quantitative\ntool for predicting and deconvoluting carbohydrate–protein\ninteractions, with potential application to design of therapeutics,\nvaccines, and diagnostics.