Probing the sialic acid binding site of the hemagglutinin-neuraminidase of Newcastle disease virus: identification of key amino acids involved in cell binding, catalysis, and fusion

Abstract

We recently reported the first crystal structure of a paramyxovirus hemagglutinin-neuraminidase (HN) from Newcastle disease virus. This multifunctional protein is responsible for binding to cellular sialyl-glycoconjugate receptors, promotion of fusion through interaction with the second viral surface fusion (F) glycoprotein, and processing progeny virions by removal of sialic acid from newly synthesized viral coat proteins. Our structural studies suggest that HN possesses a single sialic acid recognition site that can be switched between being a binding site and a catalytic site. Here we examine the effect of mutation of several conserved amino acids around the binding site on the hemagglutination, neuraminidase, and fusion functions of HN. Most mutations around the binding site result in loss of neuraminidase activity, whereas the effect on receptor binding is more variable. Residues E401, R416, and Y526 appear to be key for receptor binding. The increase in fusion promotion seen in some mutants that lack receptor binding activity presents a conundrum. We propose that in these cases HN may be switched into a fusion-promoting state through a series of conformational changes that propagate from the sialic acid binding site through to the HN dimer interface. These results further support the single-site model and suggest certain residues to be important for the triggering of fusion.

FIG. 1.

The sialic acid binding site of NDV HN. Hydrogen bonding interactions are shown as dotted lines; water molecules are labeled W. (a) complex with Neu5Ac2en obtained at pH 6.3. (b) complex with sialic acid (in its β-anomeric form) obtained at pH 4.6.

FIG. 2.

Biological activities of active site mutants of NDV HN. Bar chart represents normalized percentage values of neuraminidase activity and HAd (HA) activity relative to percentage amount of HN expressed at the cell surface. Values shown are the mean result of three independent experiments.

FIG. 3.

Membrane fusion induced by coexpressed HN and F. HeLa T4 cells were transfected with NDV F cDNA and wild-type (WT) or mutant NDV HN cDNA and were incubated for 48 h at 37°C. Cells were stained with crystal violet.

FIG. 4.

Sialyllactose modelling. (a) Four sialyllactose ligands taken from protein-ligand complexes have been superimposed on their sialic acid moieties, shown in atom coloring: yellow carbons, blue nitrogens, and red oxygens. The galactose and glucose residues are colored grey for the sugars from their complex with the influenza virus hemagglutinin, cyan for leukoagglutinin, magenta for sialoadhesin, and green for murine polyomavirus. (b) Sialyllactose modelled into the binding site of NDV HN. The side chains of the unliganded crystallographic structure are shown in grey, and the modelled structure is shown in green.

FIG. 5.

Surface electrostatic potential around the binding site. Blue indicates positive potential; red indicates negative potential, calculated in both cases for the complete HN dimer but omitting the ligand, using GRASP. (a) Complex with Neu5Ac2en. (b) Complex with sialic acid (in its β-anomeric form).