The catalytic sites of influenza virus neuraminidase are located on the upper corners of the box-shaped tetramer that forms the head of the molecule. Antigenic determinants form a nearly-continuous surface across the top of the monomer encircling the catalytic site. Approximately the same number of amino acid sequence changes occurred in these determinants between the years 1968 and 1975 as occurred in the antigenic sites of influenza virus haemagglutinin in the same period.

The first paper in this series (see previous article) described structure-activity studies of carboxamide analogues of zanamivir binding to influenza virus sialidase types A and B and showed that inhibitory activity of these compounds was much greater against influenza A enzyme. To understand the large differences in affinities, a number of protein-ligand complexes have been investigated using crystallography and molecular dynamics. The crystallographic studies show that the binding of ligands containing tertiary amide groups is accompanied by the formation of an intramolecular planar salt bridge between two amino acid residues in the active site of the enzyme. It is proposed that the unexpected strong binding of these inhibitors is a result of the burial of hydrophobic surface area and salt-bridge formation in an environment of low dielectric. In sialidase from type A virus, binding of the carboxamide moeity and salt-bridge formation have only a minor effect on the positions of the surrounding residues, whereas in type B enzyme, significant distortion of the protein is observed. The results suggest that the decreased affinity in enzyme from influenza B is directly correlated with the small changes that occur in the amino acid residue interactions accompanying ligand binding. Molecular dynamics calculations have shown that the tendency for salt-bridge formation is greater in influenza A sialidase than influenza B sialidase and that this tendency is a useful descriptor for the prediction of inhibitor potency.

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