Role of secondary sialic acid binding sites in influenza N1 neuraminidase

Abstract

Within influenza viral particles, the intricate balance between host cell binding and sialic acid receptor destruction is carefully maintained by the hemagglutinin (HA) and neuraminidase (NA) glycoproteins, respectively. A major outstanding question in influenza biology is the function of a secondary sialic acid binding site on the NA enzyme. Through a series of Brownian dynamics (BD) simulations of the avian N1, human pandemic N2, and currently circulating pandemic (H1)N1 enzymes, we have probed the role of this secondary sialic acid binding site in the avian N1 subtype. Our results suggest that electrostatic interactions at the secondary and primary sites in avian NA may play a key role in the recognition process of the sialic acid receptors and catalytic efficiency of NA. This secondary site appears to facilitate the formation of complexes with the NA protein and the sialic acid receptors, as well as provide HA activity to a lesser extent. Moreover, this site is able to steer inhibitor binding as well, albeit with reduced capacity in N1, and may have potential implications for drug resistance or optimal inhibitor design. Although the secondary sialic acid binding site has previously been shown to be nonconserved in swine NA strains, our investigations of the currently circulating pandemic H1N1 strain of swine origin appears to have retained some of the key features of the secondary sialic acid binding site. Our results indicate possible lowered HA activity for this secondary sialic acid site, which may be an important event in the emergence of the current pandemic strain.

Figure 1

Top view of the N1 tetramer (PDB: 2HTY) depicted in solvent exposed surface area representation; active sites are shown in orange, and secondary sialic acid binding sites are in green (left panel). Electrostatic potential surfaces of the area outlined in the red box are shown in the remaining panels for avian N1 (PDB: 2HTY), human N2 (PDB: 1NN2), and the currently circulating pandemic H1N1 strain (homology model presented in ref (19)). Within these surfaces, red represents −10 kcal mol⁻¹ e⁻¹ and blue represents +10 kcal mol⁻¹ e⁻¹. Sialic acid molecules are shown as licorice bound in the active (yellow) and secondary (cyan) sites.

Figure 2

A sequence comparison of critical secondary sialic acid binding site residues in the avian N1, human N2, and currently circulating pandemic strain with possible swine origin is shown. Key conserved residues that make contact with sialic acid are underlined, as derived from ref (6).