Analysis of protein-glycan recognition using absolute binding free energies

Carbohydrates are key biological mediators of molecular recognition and signalling processes. In this work, we explore the ability of absolute binding free energy (ABFE) calculations to predict the affinities of a set of five related carbohydrate ligands for the lectin protein, concanavalin A, ranging from 27-atom monosaccharides to a 120-atom complex-type N-linked glycan core pentasaccharide. ABFE calculations quantitatively rank and estimate the affinity of the ligands in relation to microcalorimetry, with a mean signed error in binding free energy of -0.63 ± 0.04 kcal/mol. Consequently, the diminished binding efficiencies of the larger carbohydrate ligands are closely reproduced: the ligand efficiency values from isothermal titration calorimetry for the glycan core pentasaccharide and its constituent trisaccharide and monosaccharide compounds are respectively -0.14 ± 0.00, -0.22 ± 0.00 and -0.41 ± 0.00 kcal/mol per heavy atom. ABFE calculations predict these ligand efficiencies to be -0.14 ± 0.02, -0.24 ± 0.03 and -0.46 ± 0.06 kcal/mol per heavy atom respectively. Consequently, the ABFE method correctly identifies the high affinity of the key anchoring mannose residue and the negligible contribution to binding of both β-GlcNAc arms of the pentasaccharide. While challenges remain in sampling the conformation and interactions of these polar, flexible and weakly bound ligands, we nevertheless here find that the ABFE method performs well. The method shows excellent promise as a quantitative tool for predicting and deconvoluting carbohydrate-protein interactions, with potential application to design of therapeutics, vaccines and diagnostics.