Avian Receptor Binding by H5 Avian Virus HA.

The H5 avian HA, which contains Gln-226/Gly-228 and prefers α2,3 linkages (25), binds to α2,3 LSTa (Fig. ​(Fig.22 a and b) through hydrogen bonds to the glycosidic oxygen of the linkage and the axial, 4-OH of Gal-2 to the amine and carbonyl groups of Gln-226 (dotted lines in Fig. ​Fig.22c). The glycosidic oxygen is exposed to Gln-226 because LSTa binds in a trans conformation (Fig. ​(Fig.2c2c Inset) around the bond between the C2 and glycosidic oxygen (star in Fig. ​Fig.22c). The trans conformation projects the Gal-2 ring up and out of the site (bold arrow in Fig. ​Fig.22c), away from the protein surface. The orientation of the Gal-2 ring (edge-on in Fig. ​Fig.22c) around the bond from the glycosidic oxygen (star in Fig. ​Fig.22c) to the C3 atom of galactose is determined by steric factors within the ligand, which avoid interactions between the 3- and 4-OH groups of Gal-2 and the ring of sialic acid. GlcNAc-3 is positioned outside and in front of the binding pocket and makes no contacts with the protein (Fig. ​(Fig.22c). Gal-4 and Glc-5 are not visible in the electron density map.

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Figure 2

The interaction of H5 and H9 influenza virus subtype HAs with avian α2,3 and human α2,6 sialosides. (a and b) Chemical formula of the pentasaccharides LSTa and LSTc. (c) Avian H5 HA with LSTa (α2,3) pentasaccharide bound. The α2,3 linkage is in trans conformation (bold arrow points up, 180° from the bond to the carboxylate of Sia-1). Hydrogen bonds (dashed lines) from Gln-226 to the α2,3-specific motif [4-OH of Gal-1 and glycosidic-O (at *) of trans linkage]. (d) Avian H5 HA with LSTc (α2,6) pentasaccharide bound. Only sialic acid of the ligand is ordered. (e) Swine H9 HA with LSTa (α2,3) pentasaccharide bound. Only two saccharides are ordered. The bound α2,3 linkage conformation is in cis conformation [bold arrow points nearly horizontal, 60° from the bond to the carboxylate of Sia-1; compare with bold arrow (trans) in c]. Contacts and hydrogen bonds (dotted lines) to Leu-226 and the carbonyl-O of residue 225 are shown. (f) Swine H9 HA with LSTc (α2,6) pentasaccharide bound. All five saccharides are ordered. The α2,6 linkage is in a cis conformation (bold arrow points nearly horizontal).

Thus, H5 avian HA contacts only the sialic acid, the α2,3-linkage glycosidic oxygen, and the axial 4-OH of Gal-2. The trans conformation of the α2,3 glycosidic linkage forms a motif of the glycosidic oxygen and the axial 4-OH of Gal-2 that is complementary to the hydrogen-bonding capacity of Gln-226 (Fig. ​(Fig.22c). [Both cis and trans are low-energy conformations of α2,3 linkages, and both have been observed in studies of sialosides in solution and bound to other proteins (26–31).]

Human Receptor Binding by H5 Avian Virus HA.

Electron density for α2,6 LSTc bound to the H5 avian HA (the nonpreferred combination) shows a poorly defined sialic acid moiety: three segments of density are visible, representing C2 and the attached carboxylate group, C5 and its N-acetyl substituent, and C6 with the glycerol group (Fig. ​(Fig.22d). The remaining four residues of the pentasaccharide are not visible. The quality of the x-ray data (better R_sym than the α2,3 data: 0.057 vs. 0.106) and the statistics from the crystallographic refinement (better R_free than the α2,3 model: 0.22 vs. 0.240) suggest that the poor quality of the image of the α2,6 ligand indicates low affinity binding and/or the binding of multiple conformations.

In solution, α2,6 linkages are predominantly in a cis conformation, with a cis-to-trans ratio of 9:1 (27) (for cis definition, see Fig. ​Fig.22e). If LSTc bound in a cis conformation to the H5 HA, then the Gal-2 C6 atom would cover Gln-226 (number 6 in Fig. ​Fig.22d), shielding it from making hydrogen bonds, and the glycosidic oxygen (* in Fig. ​Fig.22d) would be exposed to solvent (upward in Fig. ​Fig.22 c– f). If LSTc bound in a trans conformation, the glycosidic oxygen may hydrogen-bond to Gln-226, but the 4-OH of Gal-2 would be too far away to mimic the α2,3-specific motif, and there would be an energetic penalty for choosing the less stable conformer.

Avian and Human Receptor Binding by H9 Swine Virus HA.

The H9 swine virus HA, which contains Leu-226 and Gly-228, interacts with both α2,3 LSTa and α2,6 LSTc by van der Wall contacts with a methyl group of Leu-226 (Fig. ​(Fig.22 e and f). Both ligands bind to H9 HA in a cis conformation (Fig. ​(Fig.2e 2e Inset) around the bond between Sia-1 C2 and the glycosidic oxygen (star in Fig. ​Fig.22 e and f), which leaves the glycosidic oxygen exposed away from the nonpolar Leu-226 and up toward solution (up in Fig. ​Fig.22 c– f), where it might hydrogen-bond with water. This cis conformation around the glycosidic bond also projects the linkage carbon atoms of Gal-2, C3 in LSTa (bold arrows in Fig. ​Fig.22e), C6 in LSTc (bold arrows in Fig. ​Fig.22f), and the Gal-2 ring of LSTa toward nonpolar contacts with Leu-226. Steric factors within the two ligands when bound in a cis conformation orient the Gal-2 ring perpendicular (face-on in Fig. ​Fig.22 e and f) to the sialic acid ring, permitting a hydrogen bond from the 6-OH of LSTa and the main-chain carbonyl oxygen of residue 225.

Only the first (distal) two saccharides are observed in the H9/α2,3 complex, suggesting that the remainder are disordered (Fig. ​(Fig.22e).

HA Contacts with the Asialo Segment of the Pentasaccharides.

In the preferred pairing, H9 with LSTc (Fig. ​(Fig.22f), the three proximal saccharides (GlcNAc-3, Gal-4, Glc-5) fold back over the top of the sialic acid and exit from the site at the front-right. Approximately 59 Ų of nonpolar surface area is buried between Sia-1 and GlcNAc-3. Gal-4 and Glc-5 also contact Sia-1 (Table ​(Table2) 2) and contact the binding site at the loop formed by residues 125–137, at the 150 loop, and at Asn-193, burying ≈201 Ų of protein surface (Fig. ​(Fig.33a). Up to 11 hydrogen bonds could form between these proximal saccharides and the HA and two with the 4-OH group of the sialic acid (Fig. ​(Fig.33a).

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Figure 3

Atomic interactions between H5, H9, and H3 HAs and sialosides. (a) Potential hydrogen bonds (dashed red) between LSTc and the swine H9 HA. (b) Comparison of the binding of LSTc (α2,6) with the 1999 swine H9 (red) and 1968 human H3 (blue) subtype HA. Purple circles, common contacts; blue, H3 contacts; red, H9 contacts. (c) Comparison of the binding of LSTa (α2,3) with the 1997 avian H5 (green), 1999 swine H9 (red), and 1968 human H3 (blue) subtype HAs. Purple circles, common contacts; blue, H3 contacts; green, H5 contacts; red, H9 contacts.

The bound pentasaccharides in the four complexes reported here (Fig. ​(Fig.2 2 c– f) are not near crystal lattice contacts (>10 Å), suggesting that the oligosaccharide conformations observed result from inherent conformational preferences within the oligosaccharides and from the contacts observed with the HA. A folded conformation for LSTc was observed earlier in the H3 HA/LSTc complex, but the ligand conformation differed in positioning the proximal three saccharides (GlcNAc-3, Gal-4, and Glc-5) toward the back and top of the binding site (blue in Fig. ​Fig.33b) (19) rather than toward the front and right as seen here (red in Fig. ​Fig.33b). The different bound conformations of LSTc, mainly a difference of about a 60^o rotation about one bond between Gal-2 and GlcNAc-3, may be due to the slightly different position of the HA main chain at residues 133–135 which, in H9 HA, position the carbonyl oxygens of 133 and 135 and the amide of 133 closer into the binding site, where they may hydrogen-bond to Glc-5 (Fig. ​(Fig.33a). Sequence differences between the H3 and H9 HAs at 135 (Thr-H9; Gly-H3) and 133 (Ser-133; Asn-H3) and the fact that Ser-133 in H9 points into the binding site whereas the H3 Asn-133 points outward contribute two more potential hydrogen bonds in the H9/LSTc case (Fig. ​(Fig.33a). Mutagenesis studies could access the contribution to the saccharide conformation of these interactions and others with residues 193, 156, and 155 (Fig. ​(Fig.33a). Another possible source of the difference in the LSTc conformations may result from the difference in the position of Gal-2 and GlcNAc-3, resulting from the sequence differences at 228 and 190 (Gly/Val-H9; Ser/Glu-H3).

The first two saccharides of LSTa (α2,3) bind almost identically to the Leu-226-containing H9 swine and H3 human virus HAs (red and blue in Fig. ​Fig.33c), although two more of the proximal saccharides were observed in the H3 crystal structure (19). In the preferred combination, LSTa with the Gln-226-containing avian H5 HA, the trans conformation at the α(2,3) linkage (green and bold arrow in Fig. ​Fig.33c) projects the proximal saccharides up and away from the 220s loop, and a contact from Gal-2 to Glu-190 is observed.

By contrast, only sialic acid and fragments of the penultimate Gal-2 are visible in electron density of the nonpreferred pairings, H5 with α2,6 LSTc (Fig. ​(Fig.22d) and H9 with α2,3 LSTa (Fig. ​(Fig.22e).

We thank members of our laboratories for helpful discussions, the beamline staff at 14BMC at APS for assistance in data collection, and Dr. Chi-Huey Wong and colleagues for a helpful synthesis. This research is supported by National Institutes of Health Grant AI-13654, by a supplement to that grant for Expanded International Research on Emerging and Re-Emerging Diseases, by an International Partnership Research Award in Veterinary Epidemiology of the Wellcome Trust, by the Howard Hughes Medical Institute, and by the Medical Research Council. Y.H. is supported by the Jane Coffin Childs Memorial Fund for Medical Research. D.C.W. is an investigator of the Howard Hughes Medical Institute.