Structure of the H5 and H9 haemagglutinins

Like the H3 HA and influenza C HEF, H5 and H9 subtype HAs are trimeric molecules in which each monomer contains two disulfide-linked polypeptide chains, HA1 and HA2, generated by proteolytic cleavage of a single chain precursor, HA0 (Figure 2A and B) (Wiley and Skehel, 1987; Skehel and Wiley, 2000). The membrane-proximal stem of the trimer is composed of HA2 (red in Figure 2) and two segments of HA1, residues 1–55 (pink in Figure 2) and 275–329 (purple in Figure 2), centred around a triple-stranded α-helical coiled coil formed by the N-terminal half of the long central α-helix of HA2 (helix B in Figure 2C). On top of the stem, the membrane-distal globular portion of the molecule contains the receptor-binding subdomain (blue in Figure 2) that includes the receptor-binding site (labelled in Figure 2C), and the vestigial fragment of an esterase subdomain (yellow in Figure 2A–E) defined previously by its close structural similarity to a portion (54%) of the 9-O-acetylesterase domain of influenza C HEF (yellow in Figure 2F) (Rosenthal et al., 1998; Zhang et al., 1999).

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Fig. 2. H5 avian and H9 swine HA structures compared with H3 HA and HEF. (A) Ribbon diagram of the trimer of H5 (A/Dk/Sing/97) avian HA coloured by subdomains: receptor subdomain R (blue), vestigial enzyme subdomain E′ (yellow), HA2 stem F subdomain (red), F′ subdomain HA1 1–52 (pink), F′ subdomain HA1 275–307 (purple). Oligosaccharides are coloured by atom type. (B) Trimer of H9 swine (A/Sw/9/98) HA. (C) Monomer of H5 (A/Dk/Sing/97) avian HA. Helix A and B, the interhelical loop between them, and the fusion peptide in HA2 are labelled. N1 and C1 indicate the termini of HA1; N2 and C2, those of HA2. The trimer axis, receptor-binding site and two prominent antigenic loops, 140s and 150s, are labelled. Residue numbers mark oligosaccharides, which are coloured by atom type. Carbohydrate attachment sites are at HA1 21, 33, 169 and 289, and at HA2 154. (D) Monomer of the H9 (A/Sw/9/98) swine HA. The x marks the deleted 140s loop. Carbohydrate attachment sites are at HA1 21, 128, 210, 289 and 296, and at HA2 154. (E) Monomer of trimeric H3 (A/Aichi/2/68) human HA (Wilson et al., 1981). Carbohydrate attachment sites are at HA1 8, 22, 38, 81, 165 and 285, and at HA2 154. (F) Monomer of trimeric influenza C virus (C/Johannesburg/1/66) haemagglutinin–esterase–fusion (HEF) protein (Rosenthal et al., 1998; Zhang et al., 1999). These figures and Figures 3B, 4A, B and D were generated using MOLSCRIPT and BOBSCRIPT (Kraulis, 1991; Esnouf, 1997).

The H5 and H9 HAs have 46% sequence identity in HA1 and 62% in HA2. They have 39 and 58%, and 39 and 54% sequence identities, respectively, with the 1968 H3 HA1 and HA2 chains (Figure 2E) and <17% identities with the influenza C virus HEF polypeptides (Figure 2F) (Influenza sequence database at Los Alamos National Laboratory; Macken et al., 2001).

As expected from the level of sequence identity, the overall secondary and tertiary structures of the H5 and H9 HAs are very similar to those of the H3 HA (Figure 2C–E). A prominent loop at HA1 residues 140–147, shown to bind antibodies and undergo antigenic variation in H3 HAs (Wiley et al., 1981), is absent from the H9 HA (x in Figure 2D).The H5 and H9 HAs share three asparagine-linked glycosylation sites (at HA1 21 and 289, and HA2 154) (Figure 2C and D), but have only the HA2 154 site, a site conserved in all known HA sequences of the 15 influenza subtypes, in common with the seven sites of the 1968 H3 HA (Figure 2E), although three pairs of sites in H5 and H3 are near each other in sequence: HA1 33 and 38, 169 and 165, and 289 and 285, respectively. HAs from viruses isolated in the 1997 H5 and 1999 H9 outbreaks in Hong Kong have novel oligosaccharide sites relative to the most closely related avian viruses (H5, HA1 158 and HA2 57; and H9, HA1 95 and 197; Figure 1). A large oligosaccharide at HA1 158 in the prominent antigenic loop at the top of the HA on the edge of the receptor-binding site (labelled 150s loop in Figure 2C) has been shown to decrease receptor binding, possibly altering the host or the cells within a host that the virus could infect (Gambaryan et al., 1998; Lu et al., 1998; Matrosovich et al., 1999; Lin et al., 2000). The novel oligosaccharide sites, in H9 human HA relative to H9 swine HA, at positions 95 and 197, may affect the antigenicity of the human HA.

The H5 human sequences also differ from the H5 avian HA studied here by the insertion of a polybasic furin cleavage site (RRRKK) between HA1 and HA2, which has been correlated in both the H5 and the H7 subtypes with increased pathogenicity (Klenk and Garten, 1994). This insertion is expected only to change the structure of the uncleaved precursor HA0 by lengthening the prominent surface loop at the cleavage site between HA1 and HA2 (between C1 and N2 in Figure 2C and D) (Chen et al., 1998).

Most avian H9 HAs, like the H5 human and avian HAs, have the common avian HA1 residues Gln226 and Gly228 in the receptor-binding site, but the swine, human and a number of avian H9 HAs have HA1 Leu226 and HA1 Gly228, intermediate between the avian sequence and the human HA1 Leu226, HA1 Ser228 pair (Lin et al., 2000; Ha et al., 2001). Two other receptor-binding site residues that are unusual in the H9 swine HA, HA1 Asn183 and Val190, are His183 and Glu190 in human and quail H9 HAs as they are in H3 human HAs (Lin et al., 2000; Ha et al., 2001). The human H9 isolates also contain HA1 His17, like H3 viruses, rather than HA1 Tyr17 as seen in the H9 swine HA studied here. Whether this substitution at HA1 17, which may influence the pH and the mechanism of fusion activation, favours infection in humans is unknown. The remaining residue differences between H5 avian and human viruses, and between H9 swine and human viruses, occur at positions on the surface of the HA, which readily accept mutations (reviewed in Skehel and Wiley, 2000) and where many differences are observed that alter antigenicity within subtypes and probably between subtypes (Nobusawa et al., 1991; Gorman et al., 1992; Webster et al., 1992).

Rotation of the membrane-distal subdomains of H5 and H9 HAs relative to those of the H3 HA

When the central triple-stranded α-helical stem of HA2 of the H5, H9 and H3 HAs are superimposed (helix B in Figure 2C–E), the membrane-distal globular domains of H5 and H9 are rotated about the trimer molecular axis through ~20° relative to H3 and displaced upwards by ~4 Å (Figure 3B and D). H5 differs from H9 by only ~5°. The three subdomains R, E′ and F′ (a two-layer β-sheet subdomain in the stem region, residues HA1 275–307) are each relocated independently by different translations (from 5 to 8 Å) and rotations (from 16 to 19°) around slightly different axes (Figure 3B). Each subdomain has apparently migrated as a rigid body because structural differences within each subdomain among the three HA subtypes are small (r.m.s.d. ~1 Å). The independent repositioning of the R and E′ subdomains is evidence of their separate identity and stability, and provides further support for the suggestion that HEF and HA derived evolutionarily from distinct ancestral protein domains with independent functions (Rosenthal et al., 1998; Zhang et al., 1999).

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Fig. 3. Differences in subdomain locations in H3, H5 and H9 HAs and schematic diagram of key residues determining HA subtype structures. (A) R.m.s.ds between the α-carbon coordinates of the H5 and H3 HAs are plotted against HA sequence number. HA1 blue line = R subdomain superposition, yellow = E subdomain superposition, red = F′ subdomain superposition. An asterisk marks insertions in H5 relative to H3 (HA1 residues 53, 81, 95, 125, 133 and 255) and + marks a deletion at HA1 124. The underlines designate interface residues (F′–E′, orange; R–E′, green; F–E′, red; F–R, red) and the subdomain locations: E′, yellow; R, blue; and F and F′, red. (B) Superposition of the long (B) α-helices of HA2 showing differences in the intrahelical loop and long (B) α-helix N-terminal conformations: H9, blue; H5, red; H3, grey. Insert: schematic diagram indicating that the H5 and H9 globular domains are rotated 20° and displaced distally by 4 Å relative to the H3 HA. Within individual subdomains, the r.m.s.d. overlaps are 1.1, 1.0 and 1.1 Å for R, E′ and F′, respectively. The interhelical loop of HA2 contacts the R, E′ and F′ subdomains of HA1 (dashed line). (C) Sequence alignment of 15 HA subtypes at HA1 107 and HA2 60–90. Residues (*) suggested to determine the conformational differences between subtypes are shaded grey. The groups of subtypes (clades) predicted to have similar structures are shaded in four colours (grey, H3/H4/H14, G-75, K-88; yellow, H7/H10/H15, non-G-75, non-K-88, non-E-107; red, H1/H2/H5/H6/H11/H13, non-G-75, non-K-88, E-107; blue, H8/H9/H12, non-G-75, non-K-88, E-107, Q-87). The strains used to represent each subtype were: H1 (A/USSR/90/77), H2 (A/RI/5+/57), H3 (A/Aichi/2/68), H4 (A/Dk/Czechoslovakia/56), H5 (A/Dk/Singapore/3/97), H6 (A/Sh/Australia/1/72), H7 (A/Eq/Prague/1/56), H8 (A/Tk/Ontario/6118/68), H9 (A/Sw/HK/9/98), H10 (A/Ck/Germany/N/49), H11 (A/Dk/England/56), H12 (A/Dk/Alberta/60/76), H13 (A/Gl/Maryland/704/77), H14 (A/Ma/Gurjev/263/82) and H15 (A/Sh/West Australia/2576/79). (D) Phylogenetic relationships between HA subtypes and influenza B (B/Lee/40) HA based on HA amino acid sequences. The displacement of the HA1 subdomains (insert: HA viewed down the trimer axis from the top; the asterisk marks the interface where H3/H9 differ from H5 near residue 216) probably occurred when the H3, H4, H14 group diverged from the H7, H10 and H15 subtypes. The shaded colours for groups of subtypes correspond to the colours and groups in (C).

The membrane-distal globular domain of HEF is rotated a further 30° relative to the H5 and H9 HAs, or ~50° relative to H3. The different angular position of the globular membrane-distal domain of HEF, relative to the HAs, apparently results from a completely different interface between this domain and the central stem of the molecule: HEF has an extra 100 residues in the esterase subdomain, E, which are located at the interface with the stem; the central long α-helices of HEF2 are two turns shorter (Figure 2F) than the homologous α-helices in HA; and these three long α-helices diverge at their N-termini, rather than being packed into a coiled coil as in the HAs.

Ionizable residues and fusion pH refolding in different HA subtypes

Mutant selection and mutagenesis studies have identified ionizable residues that affect the pH at which the structural changes in HA required for membrane fusion occur in H3 HA (Daniels et al., 1985, 1987; Doms et al., 1986; Steinhauer et al., 1996). The wide distribution of these residues in the interfaces between all the structural elements that rearrange at low pH (Bullough et al., 1994) suggested that any destabilization of these interfaces could affect the pH of refolding and that the trigger for refolding may be distributed rather than localized. Most of the residues that influence the pH of refolding, however, were found subsequently to be in the same chemical environment in the uncleaved precursor HA0, which does not undergo the low pH-induced, irreversible refolding required for fusion (Chen et al., 1998). Only three ionizable residues, His17 of HA1 and Asp109 and Asp112 of HA2, changed from solvent-accessible to completely buried environments between the uncleaved and cleaved H3 subtype structures, raising the possibility that they may have a special role in triggering refolding (Chen et al., 1998).

We have discussed before the roles of the two acidic residues HA2 Asp109 and Asp112, which are buried by the fusion peptide on cleavage of the precursor HA0 (Chen et al., 1998). Neither H5 nor H9 HAs analysed here contain a histidine at the third buried position, HA1 residue 17; in both cases, HA1 17 is tyrosine (Table I). Although this is not strictly a subtype-specific property, it is notable that among the vast majority of HA sequences, six subtypes comprising two clades have histidine at HA1 17 (H3, H4, H7, H10, H14 and H15); the other nine subtypes comprising the other two clades have HA1 Tyr17.

In the H5 HA structure, we observe that His111 of HA2 is the only solvent-inaccessible, basic residue, and that it is near the fusion peptide (arrow in Figure 4C), behind HA2 Phe110 (Figure 4C). In H5, HA2 His111 may become ionized at low pH and contribute to the triggering of refolding. HA2 111 is histidine in the nine subtypes where HA1 17 is tyrosine (Table I). In the H5 and H9 HA structures, HA2 Arg106 and HA2 Lys106, respectively, are also located in this solvent cavity with restricted access via small channels to bulk solvent (Figure 4C, green arrows in Figure 4D). These basic residues at HA2 106 are on the long α-helix, helix B of HA2, pointing toward each other and toward the HA2 trimer axis of symmetry just above the fusion peptide, at the point where the long α-helix unfolds and reverses direction at low pH (Bullough et al., 1994) (Figure 4). In H9 HA, HA2 Lys106 is hydrogen-bonded to HA2 Glu103, which in turn hydrogen-bonds to HA2 Lys51 (dashed lines in Figure 4C). There is a strong peak (>5σ) of electron density with approximately tetrahedral shape buried on the trimer axis above the three HA2 Lys106 residues in the trimer that may be an anion (possibly phosphate, with a refined temperature factor of 29.5 Å²), which could stabilize the HA2 Lys106 positive charges (blue mesh in Figure 4B). In the H5 HA, where the charge on HA2 Arg106 is apparently balanced by the salt bridge hydrogen bond to HA2 Glu105 (glutamine in H9) (but a dipole–dipole repulsion may persist), there are water molecules or monovalent ions near the trimer axis below HA2 Arg106, but there is apparently no large ion above or near the positions of the three Arg106 residues in the trimer. In the H3 HA, HA2 His106 is located in approximately the same position, probably hydrogen-bonded to HA2 Lys51 and HA2 Gln105. The two clades with HA2 His111 that lack HA1 His17 contain arginine or lysine at HA2 position 106, suggesting that if these residues prove to be important in refolding at low pH, they may have evolved as alternatives to HA1 His17 as components of the triggering mechanism.

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Fig. 4. H5 and H9 residues implicated in the low pH-induced refolding required for membrane fusion. (A) An H5 HA trimer with monomers coloured blue, pink and white. The box indicates approximately the area detailed in (B–D). Red segments of the fusion peptide and near HA1 Met30 are indicated by red dotted lines in (D). (B) H9 HA difference in electron density (contoured at 4σ) suggests an ion bound between three HA2 Lys106 residues at the centre of the triple-helical stem region in H9. HA2 Glu103 is hydrogen-bonded to both HA2 Lys51 and HA2 Lys106. A number of water molecules are found around the putative counter-ion and below HA2 Lys106 (not shown). (C) H5 HA2 His111 is buried by HA1 Leu320, HA2 Trp21 and Phe110, and is located near the fusion peptide (arrow), HA2 residues G1, L2, F3 coloured blue. (D) In the HA1–HA2 trimer, residue HA2 106 is accessible to solvent only through narrow channels (green arrows) after the fusion peptide (G1, L2 and F3) fits between the helices following HA0 cleavage to HA1 and HA2 (Chen et al., 1998). For clarity, one helix in the front of the cavity and peripheral residues were omitted. This figure was generated using GRASP (Nicholls et al., 1991).

A difference in solvent accessibility in uncleaved and cleaved HAs of the cavity near HA2 positions 111 and 106 is caused by HA2 residues Gly1, Leu2 and Phe3 of the fusion peptide, which occlude most of the channel below HA1 Met30 (Figure 4D) that connects the central cavity to the bulk solvent. It is possible that changes in ionization at low pH may be relieved, in the relatively open cavity of uncleaved HA0 near residues HA2 106 and the putative anion, by local rearrangements. However, following HA0 cleavage, when that cavity is nearly occluded from bulk solvent by the refolded fusion peptide, changes in ionization may contribute to the triggering of the structural changes in HA required for membrane fusion.

Crystal growth and ligand soaking

Hanging drop crystallizations used 1 µl of BHA, 10 mg/ml, 10 mM HEPES buffer pH 7.5 mixed with 1 µl of precipitant solution (H5, 20% MME-PEG 2000, 2 mM NiCl₂, 100 mM HEPES pH 7.5; H9, 1.4 M Na citrate, 100 mM HEPES pH 7.5). Crystals were transferred to 200 µl of well solution and then to solutions with increasing amounts of cryoprotectant in 2 h steps (H5, 20% glycerol; H9, 1.5 M xylitol). Crystals were flash-cooled in liquid nitrogen.

Data collection, structural determination and refinement

Both H5 and H9 native diffraction data were collected at beamline 14BMC at the Adanced Photon Source (Argonne National Laboratory), 100K; H9 derivative data were collected at beamline X25C at the National Synchrotron Light Source (Brookhaven National Laboratory), 100K. All data sets were processed with DENZO and SCALEPACK (Otwinowski and Minor, 1997).

Because the H9 HA trimer was located on the 3-fold symmetry axis of a P6₃ lattice, molecular replacement reduced to a single, angular parameter search of 120°. Conventional molecular replacement (MR) and the direct search with AMORE using the X31 H3 subtype HA coordinates (PDB: 1HGE) (H9 HA has 42% sequence identity with the H3 HA) produced a consistent solution. However, the MR-phased electron density map could not be traced in the membrane-distal domain due to domain motions. The MR phases were useful in finding heavy atom sites in derivatives by difference Fourier methods in combination with difference and anomalous Patterson methods. MIRAS phases calculated with MLPHARE (CCP4, 1994) were improved by solvent flattening and histogram matching using DM (CCP4, 1994). Models were built with O (Jones et al., 1991). One saccharide was visible at Asn21 and two at HA1 asparagines 128, 289 and 296, and HA2 154.

The H5 HA structure was determined by MR using H9 HA coordinates (H5 and H3 share 44% sequence identity; H5 and H9 52%). The electron density map calculated from the H9 MR phases was interpretable. Because the difference between H9 and X:31 indicated domain motions, rigid body refinement of each individual domain in the search probe, followed by solvent flipping and histogram matching in CNS (Brünger et al., 1998), was used to improve the electron density map. One saccharide was visible at HA1 Asn21 and two at HA1 asparagines 33, 169 and 289, and HA2 154. C-terminal segments after HA1 324 and HA2 160 are missing in the final model and are presumed to be disordered. One Ni (2+) ion, coordinated by two histidines (HA2 His142), joins two neighbouring molecules at the membrane-proximal end of HA in the H5 crystal lattice.

Both structures were refined with CNS (Brünger et al., 1998). Statistics are summarized in Table II. Coordinates have been deposited in the Protein Data Bank (accession Nos: H5, 1JSM; H9, 1JSD).

Table II.

Crystallographic data statistics

		H5	H9
Data collection
Resolution (Å)		40.0–1.9	30.0–1.8
Unique reflections		59 121 (3643)	91 194 (9090)
Completeness (%)		90.5 (56.4)	100.0 (100.0)
Redundancy		6	9
I/<σ>		17.5	12.9
Rmerge (%)		7.0 (20.6)	8.3 (48.2)
Refinement
Rcryst (%)		24.1 (40.4)	21.8 (32.5)
Rfree (%)		27.1 (53.2)	22.9 (33.2)
R.m.s.d. bond lengths (Å)		0.009	0.007
R.m.s.d angles (°)		1.5	1.3
B r.m.s.d. for bonded main chain and side chain atoms		0.929, 1.632	0.540, 0.540
No. of atoms
protein		3738	3725
water		357	282
carbohydrate		101	125
H9 derivatives and phasing	Pt(NH3)2Cl2	K2PtCl4	K3IrCl6
Resolution (Å)	25.0–2.6	25.0–2.6	25.0–2.7
Unique reflections	30 731	28 769	30 645
Completeness (%)	98.8	93.9	100.0
Redundancy	5	4	5
I/<σ>	15.5	14.2	15.0
Rmerge (%)	5.6	7.2	6.5
Phasing power acentric (centric)	1.45 (1.21)	1.36 (1.06)	0.90 (0.56–5 Å)
Rcullis acentric (centric, anomalous)	0.73 (0.71, 0.94)	0.77 (0.81, 0.97)	0.85 (0.87, 0.95)
Overall FOM	0.46

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H5 crystallized in space group R32 (a = 131.6 Å, c = 254.1 Å) and H9 in space group P6₃ (a = 108.5 Å, c = 149.3 Å). The statistics for the highest resolution shell of the native data set are in parentheses. Although the H5 crystals diffracted to high resolution, they have high mosaicity (>0.8).