Hemagglutinins from two influenza virus variants bind to sialic acid derivatives with millimolar dissociation constants: a 500-MHz proton nuclear magnetic resonance study

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Abstract
The equilibrium binding of influenza virus hemagglutinin to derivatives of its cell-surface ligand, sialic acid, was measured by nuclear magnetic resonance (NMR) spectroscooy. Binding was quantified by observing perturbations of sialic acid resonances in the presence of protein. The major pertubation observed was a chemical shift of the N-acetyl methyl resonance, persumably due to the proximity of the methyl gorup to tryptophan 153. X-31 hemagglutinin binds to the methyl .alpha.-glycoside of sialic acid with a dissociation constant of 2.8 mM and does not bind to the methyl .beta.-glycoside. Replacing the 4-hydroxyl group of sialic acid with an acetyl group has little effect, while replacing the 7-hydroxyl group with an acetyl prevents binding. Experiments with sialylated oligosaccharides confirm literature reports that mutations at amino acid 226 change the specificity of hemagglutinin for .alpha.(2,6) and .alpha.(2,3) glycosidic linkages. The NMR line broadening of sialyloligosaccharides suggests that sialic acid is the only component that contacts the protein. Saccharides containing two sialic acid residues appear to hve two separate binding modes. Hemagglutinin that has undergone a low pH induced conformational change retains the ability to bind sialic acid.