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. 2009 Oct 6;106(40):17175–17180. doi: 10.1073/pnas.0906849106

Structures of receptor complexes formed by hemagglutinins from the Asian Influenza pandemic of 1957

Junfeng Liu a, David J Stevens a, Lesley F Haire a, Philip A Walker a, Peter J Coombs a, Rupert J Russell b, Steven J Gamblin a,1, John J Skehel a,1
PMCID: PMC2761367  PMID: 19805083

Abstract

The viruses that caused the three influenza pandemics of the twentieth century in 1918, 1957, and 1968 had distinct hemagglutinin receptor binding glycoproteins that had evolved the capacity to recognize human cell receptors. We have determined the structure of the H2 hemagglutinin from the second pandemic, the “Asian Influenza” of 1957. We compare it with the 1918 “Spanish Influenza” hemagglutinin, H1, and the 1968 “Hong Kong Influenza” hemagglutinin, H3, and show that despite its close overall structural similarity to H1, and its more distant relationship to H3, the H2 receptor binding site is closely related to that of H3 hemagglutinin. By analyzing hemagglutinins of potential H2 avian precursors of the pandemic virus, we show that the human receptor can be bound by avian hemagglutinins that lack the human–specific mutations of H2 and H3 pandemic viruses, Gln-226Leu, and Gly-228Ser. We show how Gln-226 in the avian H2 receptor binding site, together with Asn-186, form hydrogen bond networks through bound water molecules to mediate binding to human receptor. We show that the human receptor adopts a very similar conformation in both human and avian hemagglutinin-receptor complexes. We also show that Leu-226 in the receptor binding site of human virus hemagglutinins creates a hydrophobic environment near the Sia-1-Gal-2 glycosidic linkage that favors binding of the human receptor and is unfavorable for avian receptor binding. We consider the significance for the development of pandemics, of the existence of avian viruses that can bind to both avian and human receptors.

The second influenza pandemic of the 20th century, “Asian Influenza,” began in 1957 (1–3) and ended with the appearance of “Hong Kong Influenza,” in 1968. The viruses responsible for the 1957 pandemic were formed by reassortment between human and avian viruses (4, 5) and belong to the H2N2 subtype, signifying that, by comparison with the H1N1 viruses of the 1918 “Spanish Influenza,” the virus surface glycoproteins, hemagglutinin (‘H’) and neuraminidase (‘N′), were antigenically different (6). Both of these glycoproteins, and the polymerase component PB1, were derived from avian viruses (4, 5). Two groups of H2 avian hemagglutinins (HA) that have been characterized by genetic and antigenic analyses, are components of viruses located either in Europe and Asia or in North America (7, 8). The HA of the 1957 pandemic is a member of the Eurasian group.

Hemagglutinin is the receptor binding glycoprotein of influenza viruses (9). The receptors are sialic acids (10) that are terminal residues of carbohydrate side-chains of cellular glycoproteins and glycolipids, and receptor recognition has been shown to vary in specificity depending on the species infected (11, 12). The focus of this specificity is the nature of the glycosidic linkage between sialic acid and the penultimate sugar of the side-chains (11, 13). HAs of avian viruses prefer sialic acid in α2,3-linkage to galactose, the avian receptor, which predominates in the avian enteric tract (14, 15), while those of human viruses prefer sialic acid in α2,6-linkage, the human receptor, which is predominant in the upper respiratory tract (16–18). The avian origin of pandemic influenza virus HAs, therefore, implies a change in receptor binding specificity. Genetic analyses have indicated that for “Asian” and “Hong Kong” pandemic viruses, the same amino acid substitutions in the receptor binding sites of HA correlate with their acquired preference for the human receptor; Gln-226Leu and Gly-228Ser (12). The three sites for sialic acid binding are at the membrane-distal tips of the identical monomers that form the HA trimer (9) (Fig. 1). Each site comprises a pocket of conserved amino acids that is edged by the membrane-distal 190-helix at the top of the site, and the 130- and 220-loops located at the front edge and the left side of the site, respectively (9, 19, 20). Thus the 226/228 pair of substitutions are located at the bottom left of the receptor binding site.

Fig. 1.

Fig. 1.

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Ribbons representation of different H2 HA monomers and receptor binding sites. (A) Superposition of the monomers of two human H2 HAs: A/Singapore/1/57 and A/Japan/305/57 colored green and yellow respectively. (B) Three avian H2 HAs: A/ck/New York/29878/91 colored gray, A/dk/Ontario/77 colored in blue and A/ck/potsdam/4705/84 colored orange red. (C) Overlap of monomers of a human H2 HA colored in green and avian H2 HA colored in blue. The region highlighted by the gray ellipse at the top of the panel shows the receptor binding domain, an expanded version of which is shown in (D). Conserved residues such as Tyr-98, Ser-136, Trp-153, and His-183 are shown in stick representation together with other residues important in receptor binding specificity such as Asn-186, Glu-190, and Leu-194, as well as the Gln/Leu-226, Gly/Ser-228 pair.

To examine the structural significance of these changes we have determined by X-ray crystallography the structures of the HAs of human and avian H2 viruses complexed with sialopentasaccharide analogues of human and avian receptors. The HAs chosen for study were from the two prototype human viruses of 1957, A/Singapore/1/57 (Singapore) (1) and A/Japan/305/57 (Japan) (2) and from one Eurasian A/ck/Potsdam/84 and two North American avian viruses, A/dk/Ontario/77 and A/ck/New York/91 (7, 8). Our results for the HA of the 1957 pandemic are compared with others derived from studies of HAs of the 1918 and 1968 pandemics, reported here and before, (21–23). To facilitate this comparison we have adopted an ‘H3’ numbering scheme for the H2 HAs.

Results

Structure Determination.

Diffraction datasets were collected from crystals of two human and three avian H2 HAs, both from native, as well as human or avian receptor sialopentasaccharide analogue-soaked, crystals (summarized in Table 1). The structures were solved by molecular replacement and subsequently refined by automated methods interspersed with manual rebuilding using COOT (24). Relevant crystallographic statistics are given in the SI.

Table 1.

Summary of the data resolution and PDB accession codes for each of the structures described in the text; full crystallographic statistics are given in the SI

HA subtype
H2 H1
Species Avian Human Avian Human
Virus New York Ontario Potsdam Singapore Japan Alberta 1918
Uncomplexed 2.4 Å 2WR0 2.6 Å 2WR5 – 2.7 Å 2WRC 3.0 Å 2WRD – –
Avian receptor 2.4 Å 2WR2 2.5 Å 2WR3 – 3.1 Å 2WRB – – –
Human receptor 2.1 Å 2WR1 2.5 Å 2WR4 3.1 Å 2WRF 2.5 Å 2WR7 3.0 Å 2WRE 3.0 Å 2WRH 3.0 Å 2WRG
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As seen for other HA subtypes, (21, 25) there is a close correspondence in the structures of H2 HA from human and avian viruses reflecting the high level of sequence identity between them (about 90%) (Fig. 1C). As expected from phylogenetic clustering (26), H2 HAs are most closely related in overall structure to H1 with root mean square deviation (r.m.s.d.) in Cα positions of about 1.3Å between monomers, in both cases. Thus the H1 and H2 HAs associated with the first two pandemic viruses in the last century are closely related but quite distinct from the third, the H3 HA of the 1968 pandemic virus. The r.m.s.d. (on all Cαs) between avian H2 and human H3 HA is 2.4Å (22), reflecting the lower sequence identity between these proteins (about 40%). This comparison is of special interest because, as mentioned above, H2 and H3 HAs share the same avian to human host adaptation mutations, Gln-226Leu and Gly-228Ser, but H1 does not (27).

Avian H2 Complexes with Avian and Human Receptors.

Avian HA with avian receptor.

The complex between avian H2 HA and an avian receptor analogue reveals that the ligand binds in a similar manner to that seen in nearly all other avian HAs, with the sialopentasaccharide adopting a trans conformation about the glycosidic bond between Sia-1 and Gal-2 (Fig. 2A) (21, 22, 25, 28). Consequently, the trajectory of the second and third sugar rings, relative to the sialic acid, is also similar to other avian HA/avian receptor complexes (Fig. 2A) (21, 25, 28). The key interactions involved in avian receptor binding are between the side-chain of the ‘avian-signature’ residue Gln-226 (28, 29) and the glycosidic oxygen between Sia-1 and Gal-2, and the 4′OH of Gal-2.

Fig. 2.

Fig. 2.

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Interactions of an avian H2 HA (upper panels) and a human H2 HA (lower panels) with avian and human receptor analogues. The three secondary structure elements of the binding site, the 130- and 220-loops and the 190-helix are labeled in this backbone representation together with some selected side chains in stick representation. The broken lines indicate potential hydrogen bond interaction. In all four panels, the sialosaccharides are colored yellow for carbon atoms, blue for nitrogen, and red for oxygen, water molecules are indicated by red spheres. A/dk/Ontario/77 H2 HA, colored blue, in complex with avian receptor, LSTa, (A) and human receptor, LSTc. (B). A/Singapore/1/57 H2 HA, colored in green, in complex with human receptor (C) and avian receptor (D). The black arrows in A, B, and C indicate that for the two human receptor complexes the Sia-1/Gal-2 linkage adopts a cis conformation, whereas for the avian complex it adopts a trans conformation.

Avian haemagglutinin with human receptor.

The structure of avian H2 in complex with human receptor (Fig. 2B), also shows strong electron density extending to at least the third sugar, GlcNAc-3, of the sialopentasaccharide, which has a similar conformation to that observed for this ligand bound to human H2 HA (Fig. 2C). The Sia-1-Gal-2 glycosidic bond adopts a cis conformation and the pentasaccharide exits the binding site in approximately the opposite direction to the avian receptor (Fig. 2A). There are extensive hydrogen bond interactions between avian HA and Gal-2 of the human receptor; particularly noteworthy are the roles of two water molecules in mediating these interactions. Wat-1 links the 3′ OH of Gal-2 with the side-chain of Lys-222 and the main-chain carbonyl at 225, while Wat-2 hydrogen bonds to Gln-226 and Asn-186, as well as to the 4′OH of Gal-2 and the 9′OH of Sia-1 (Fig. 2B).

Human H2 Complexes with Human and Avian Receptors.

Human HA with human receptor.

The complex between human H2 HA and the human receptor (Fig. 2C), shows that the ligand adopts a cis conformation at the glycosidic bond between Sia-1 and Gal-2 and an overall orientation similar to that seen in complexes with human H3 haemagglutinin (Fig. 3A) (22). There is a hydrogen bond between the 3′OH of Gal-2 and the side-chain of Lys-222 (Fig. 2C), but there is no electron density for water molecules that could mediate further hydrogen bond interactions between the HA and ligand. The leucine residue at position 226 leads to a more hydrophobic environment than that present in avian HAs and likely accounts for the absence of water molecules in this region. Further, the shortest distance between the side-chain of Leu-226 and C6 of the ligand is about 4.2 Å, which is too long to generate a significant hydrophobic interaction to favor the binding of the α2,6-linked ligand. By contrast, in unliganded avian H2 HA Gln-226 coordinates a water molecule that is very close to the position occupied by Gal-2 in the human HA-human receptor complex. This suggests that human HAs benefit from acquiring leucine at position 226 because human receptors can then bind without the need to displace water.

Fig. 3.

Fig. 3.

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Overlap of the receptor binding domains of H1, H2, and H3 HAs in complexes with receptor analogues. (A) The overlapped receptor binding sites of human HAs for H1 A/S.Carolina/1918, (blue), H2 A/Singapore/1/57, (yellow) and H3 A/Aichi/2/68, (22) (gray) in complex with human receptor analogue, LSTc. (B) The receptor binding domains of avian HAs for H1, A/dk/Alberta/76 (blue), H2, A/dk/Ontario/77 (yellow), and H3, A/dk/Ukraine/63 (25) (gray) in complex with human receptor analogue. The sialopentasaccharides are colored according to the HAs to which they are bound and some of the side-chains discussed in the text are shown in stick representation.

Human HA with avian receptor.

In the complex between human H2 HA and the avian receptor analogue, there is relatively poor electron density for just the sialic acid of the receptor, indicative of weak binding to this ligand (Fig. 2D). The most obvious explanation for the poorer binding of avian receptor, that was also observed in binding assays (30), lies in the positioning of the hydrophobic leucine residue 226, underneath the position that the glycosidic oxygen would occupy in the α 2,3-linked avian receptor. A significant consequence of the fact that avian α 2,3-linked receptors tend to bind in a trans conformation at the Sia-1-Gal-2 glycosidic bond, whereas human α 2,6-linked receptors adopt a cis conformation, is that one of the loan pairs of electrons of the glycosidic oxygen in the avian receptor is oriented toward Gln-226 rather than away from the protein toward solvent, as in the case of the human receptor. Leu-226 in human H2 is, therefore, a contributory factor to the weak binding of avian receptor and this may be relevant to human adaptation since mucins in the human lung, which contain α 2,3-linked sialic acid, might block infection by viruses that bind avian receptor more strongly (17).

Overall, the structures of the H2 HA-receptor analogue complexes indicate how the avian H2 HA binds both human and avian receptors and how the human HA binds human, but not avian receptors. Prominent among the reasons for the last property is the human HA-specific amino acid substitution Gln-226Leu, which creates a hydrophobic environment in the position that would be occupied by the Sia-1-Gal-2 glycosidic linkage of an avian receptor.

Avian H1 HA in complex with human receptor.

The structures of the H3 HAs of the 1968 “Hong Kong Influenza” pandemic virus and of a possible precursor avian virus, and the HI HA of the 1918 “Spanish Influenza” pandemic virus, were determined before (21, 23, 25). To be able to compare the HAs of all three pandemic viruses, and especially because of the close similarity between the structures of H2 and H1 HAs, we have determined the structure of a 1918 human H1 HA (Fig. 3A) and of an avian H1 HA, (Fig. 3B) in complexes with a human receptor analogue. The receptor binding domain of the avian H1 is almost identical to that of the avian H2 HA. However, the electron density for the human receptor in the avian H1 complex is much poorer than in the avian H2 complex, with only part of the Gal-2 ring defined, (Fig. 3B). At the primary structure level, the only difference between the H2 and H1 HAs in this region is at position 186. Asn-186 in avian H2 which, as described above, and shown in Fig. 2B, is an important component of the hydrogen bonding network with human receptor, is replaced by a proline residue in avian H1. It seems, therefore, that this difference is, at least in part, responsible for the relatively poor binding of the human receptor by this avian H1 HA. H1 viruses isolated from humans before 2000 mainly contained Pro-186Ser mutations and were observed to favor human receptor binding (31) but since then, Pro-186 variants have predominated in human infections. The HA of the new swine HIN1 pandemic virus, however, has Serine at position 186.

Discussion

The overall structures of H2 HAs, and the close similarity with H1 and H5 HAs (21, 28) conform to the expected features for members of phylogenetic Group-1 (26) to which they belong. The structures of the human H2 HAs also allows location of previously deduced antigenically significant amino acids near residues 137, 189, 140, 200, and 214 (7, 8) and all five H2 HA structures confirm the group-specific identity and positions of residues near the “fusion peptide” that are involved in membrane fusion activation (26). Our focus here, on the receptor binding properties of the HAs of the prototype 1957 viruses and of their potential avian precursors, reflects the importance of HA-receptor interactions in the initial stages of infection and in interspecies transmission. Having 1918 H1 and 1957 H2 HA-receptor analogue complexes to complement those available for 1968 H3 HA (22, 25), enables comparison of the receptor binding sites and of the basis for the receptor binding specificity of the HAs of all three pandemic viruses.

On the basis of amino acid sequence and structure, H2 HA is very similar to H1 (21) but quite distinct from H3 (23). However, comparison of the complexes formed between human H1, H2, and H3 HAs with a human receptor shows that the H2 binding mode is much more similar to H3 than H1. Two main features distinguish the complexes: the lower position of Gal-2 in the H1 binding site, and the different orientation of the third, fourth, and fifth residues of the bound sialopentasaccharide, which is almost parallel with the 3-fold axis of symmetry of the H1 HA, compared with a more folded-back conformation in the H2 and H3 sites (Fig. 3A). Human H2 and H3 HAs share the amino acid substitutions Gln-226Leu and Gly-228Ser by comparison with avian HAs, and with human H1 HA, which retains the avian Gln-226/Gly-228 sequence. The differences in receptor positioning might, therefore, be a direct consequence of these differences in sequence. However, examination of complexes formed by avian H2 HA with human receptor (Fig. 3B) suggests an alternative. Despite the characteristic avian Gln-226/Gly-228 sequence in the avian H2 HA binding site, the conformation adopted by the human receptor is the same as that in the human H2 HA human receptor complex. The positioning of the receptor, therefore, appears not to be related directly to the Gln-226/Gly-228 sequence but to an alternative feature of the site.

The avian H2 HA-human receptor complex is remarkably well-ordered by comparison with equivalent avian H1 and avian H3 complexes. The protein environment near Gal-2 is quite similar in all three cases but it is only in H2 that there is well-defined electron density for the second and third sugars of the human receptor analogue. The most significant sequence differences that might be involved in this feature appear to be at residue 186, which is an asparagine in H2, proline in H1, and serine in H3. The role of Asn-186 in contacting Gln-226 and the 4′ OH of Gal-2 through a water molecule, is illustrated in Fig. 2B, and it is likely that the absence of this residue and of the hydrogen-bonded network that involves Gln-226, contributes to the relatively poor binding of human receptor by these avian H1 and H3 HAs. Interestingly, Asn-186 does not play this role in human H2 binding to human receptor. In that case the Gln-226Leu substitution means that the hydrogen bond network involving Asn-186 is not formed. Instead the Asn-186 side-chain points away from the receptor binding site, adopting the same orientation as in the unliganded structure.

The significance of the ability of HAs to bind both avian and human receptors has been the subject of debate, particularly concerning whether mixtures of human and avian viruses have been isolated (17, 32) or whether modifications have occurred during adaptation to growth in the laboratory in avian or mammalian cells (11, 17). Nevertheless, it would appear to be a clear advantage for an avian virus, with the overall genetic potential to cause a pandemic, to be able to bind to human receptors like the H2 avian HA described here, to gain an initial foothold for infection of humans. Under selective pressure the subsequent Gln-226Leu mutation would be expected to increase affinity for the human receptor and decrease affinity for the avian receptor (Fig. 2 C and D). The latter effect would be an advantage in avoiding interaction with avian receptor-rich, respiratory-tract mucins that would otherwise block infection (17). Early in H1, H2, and H3 pandemics, single receptor binding site mutants were detected, Gln-226Leu for H2 and H3 and Glu-190Asp for H1 (30, 33). The additional characteristic mutations Gly-228Ser in H2 and H3 and Gly-225Asp in H1 seem to have more limited effects on affinity and specificity and were presumably secondary acquisitions (30). It is also feasible that some avian viruses containing H1, H2, and H3 HAs have a particular propensity, compared with those containing HAs of other subtypes, for trans-species infection. Consistent with this possibility, there have been suggestions of “recycling” of these three subtypes in humans: in 1890 and 1968 for H3, and 1918, 1977, and 2009 for H1 viruses, although the proposal for an H2 epidemic at the end of the nineteenth century, as well as in 1957 (34) appears less likely (35). At this stage it could be informative, not least with regard to pandemic planning, to extend investigations of this possibility, (12, 31, 36, 37) using robust assays of binding specificity on a substantial number of avian viruses from all 16 HA subtypes, to see if there are other subtypes that have representatives with the ability to bind human receptors.

Methods

All HAs, except the 1918 H1 human, were from viruses grown in hens' eggs and prepared by bromelain digestion, as previously described (28). The gene for the H1 human 1918 HA was made synthetically (38) and the protein expressed in recombinant vaccinia virus infected CV1 cells, as previously described (39) and isolated by bromelain digestion. Crystallization conditions were screened by the sitting-drop vapor diffusion method using Crystal Clear strips (Douglas Instruments). The nano-drops were set up with 0.1 μL of BHA protein solution (10 mg/mL in 10 mM Tris·HCl, pH 8.0) and 0.1 μL of reservoir solution by using an Oryx-8 robot (Douglas Instruments). Crystals were grown at 18 °C using the following reservoir solutions; H2 human Singapore 32% PEG 400, 2% PEG3350, 0.1M Mes pH6.5; H2 human Japan 30% PEG 400, 0.1M Mes pH6.0; H2 avian duck Ontario 30 PEG 2K MME, 0.1M Bis Tris propane at pH 6.5; H2 avian New York 25% PEG 3350, 0.2M Ammonium acetate, 0.1M HEPES at pH 6.8; H2 avian Potsdam 22% PEG 3350, 0.1M Tris at pH 8.0; H1 avian 1976 2.3 M AS, 1% PEG 400, 0.1M Pipes pH 7.0; H1 human 1918 2.2M Ammonium sulfate, 1% PEG 400, 0.1M Bis Tris propane pH 5.5. Crystals were prepared for flash cooling by serial transfer into reservoir solution augmented by 20–25% PEG400. Crystals for datasets with bound receptor were prepared by soaking the crystals over night in either 4 mM LSTa (α2,3-linked galactose) or 4 mM LSTc (α 2,6-linked galactose) made up in cryo buffer. All crystals were screened on a Raxis4 detector (100-mm scan) mounted on a Rigaku MicroMax 007 HF generator. Ligand soaked crystals of H1 avian 1976 HA were collected at Daresbury SRS stations 14.2 and 9.6 using ADSC Quantum Q4 detectors. All of the other data were collected on beamlines IO3/IO4 at the Diamond Light Source at 100K with an ADSC Q315. Diffraction data were integrated using Denzo and scaled with Scalepack (40). Ellipsoidal Truncation and Anisotropic Scaling are performed using the server: http://www.doe-mbi.ucla.edu/∼sawaya/anisoscale/ (41). H2 A/dk/ Ontario/77 HA was solved by molecular replacement with Phaser (42) using H1, A/PR/8/34 HA, (21). All other H2 HAs were solved using H2 A/dk/ Ontario/77 HA as the search model. Standard refinement, with Refmac (43) and PHENIX (44), and manual model building with Coot (24), was performed on all of the structures. Crystallographic statistics are given in the SI, together with the relevant PDB accession codes. Molecular figures were created with Pymol (http://pymol.sourceforge.net/; 45).

Acknowledgments.

Research at the National Institute for Medical Research is funded by the Medical Research Council (U.K.). R.J.R. thanks the Scottish Funding Council. We thank R.G. Webster of St Jude Research Institute, Memphis for seeds of the avian H2 viruses and the World Health Organization Collaborating Centre for Influenza Reference and Research at National Institute for Medical Research for HA sequences.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 2WR0–2WR5, 2WR7, 2WRB–2WRH).

This article contains supporting information online at www.pnas.org/cgi/content/full/0906849106/DCSupplemental.

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