Intermolecular nuclear Overhauser effect and atomic pair potential approaches to wheat germ agglutinin-sugar binding

The detailed binding mechanism of wheat germ agglutinin (WGA) with N-acetylglucosamine (GlcNAc) was investigated using intermolecular 1H-1H nuclear Overhauser effect (NOE) and atomic pair potential (APP) calculations. Negative NOE was observed on the 1H spectrum of 1-O-methyl derivative of GlcNAc in a solution containing WGA, when the aromatic region of the WGA spectrum was irradiated. Analyses of the time dependence of NOE revealed that H2 and the N-acetyl methyl protons of the sugar are in close proximity to the aromatic protons of WGA in the bound state. This was confirmed and further elucidated by the APP calculations. According to the calculation, the major binding force comes from a hydrogen-bonding between C3-OH of sugar and an acidic residue present in each of the two binding sites of WGA: Glu115 in site 1 and Asp29 in site 2. The binding is further assisted by the N-acetyl group which interacts with a few more polar amino acid residues in the binding sites. The optimized binding mode suggested by the APP calculations supports the NMR results in that H2 and a part of the N-acetyl methyl protons are within 4.5 A distance from protons of both Tyr64 and Tyr73 in site 1 and of Tyr159 in site 2.