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. 1997 Oct 28;94(22):11808–11812. doi: 10.1073/pnas.94.22.11808

Structural evidence for a second sialic acid binding site in avian influenza virus neuraminidases

Joseph N Varghese 1,*, Peter M Colman 1, Albertus van Donkelaar 1, Tony J Blick 1, Anjali Sahasrabudhe 1, Jennifer L McKimm-Breschkin 1
PMCID: PMC23599  PMID: 9342319

Abstract

The x-ray structure of a complex of sialic acid (Neu5Ac) with neuraminidase N9 subtype from A/tern/Australia/G70C/75 influenza virus at 4°C has revealed the location of a second Neu5Ac binding site on the surface of the enzyme. At 18°C, only the enzyme active site contains bound Neu5Ac. Neu5Ac binds in the second site in the chair conformation in a similar way to which it binds to hemagglutinin. The residues that interact with Neu5Ac at this second site are mostly conserved in avian strains, but not in human and swine strains, indicating that it has some as-yet-unknown biological function in birds.

Influenza, an orthomyxo virus, has a negative-stranded RNA segmented genome that codes for two surface glycoproteins (1). One of the genes codes for neuraminidase (NA) (2), a glycoprotein found on the surface of the influenza virus particle. The NA is an enzyme that cleaves terminal sialic acid (Neu5Ac) from glycoconjugates found on the surface of molecules on target cells in the upper respiratory tract of some susceptible mammals, including humans (3, 4). These molecules with terminal Neu5Ac are also the target receptors for the viral hemagglutinin (HA) (5), the major surface glycoprotein on the viral particle surface. NA destroys these HA receptors, allowing progeny virus particles, budding from infected cell surfaces, to be released (6). It also is thought that NA facilitates passage of virus through the protective mucin covering target cells by desialylation of the Neu5Ac-rich mucin (7) and prevents aggregation by HA of freshly synthesized viral glycoproteins via sialylated carbohydrates.

The x-ray structure of influenza virus NA has been determined (8, 9) for type A subtype N2 (10), N9 (11, 12), and type B (13), together with their complexes with Neu5Ac (14) and other NA inhibitors (15–17). These structural studies have led to a renewed interest in NA inhibitors as a means of controlling influenza virus infections. Potent NA inhibitors now have been developed (15, 18, 19), and one of them, 4-guanidino-Neu5Ac2en (GG167, Zanamivir) (15, 20), now is undergoing phase III clinical trials.

HA activity has been reported for the N9 subtype of NA of influenza type A virus (21) at 4°C, which was not related to aberrant NA activity, but was associated with a second Neu5Ac binding site on the surface of NA away from the active site (22). The residues on the surface of NA responsible for the hemabsorbing (HB) activity have been identified by monoclonal variants that lost capacity to bind red blood cells (22), and the activity has been successfully transferred to the N2 subtype of NA (23) by site-directed mutagenesis. Furthermore, it was shown that N9 NA activity did not remove the putative Neu5Ac-related moiety that bound red blood cells to this HB site (24) as the agglutination was restored on cooling to 4°C. An HB site also has been discovered on the NA of A/FPV/Rostock/34 H7N1 (25), which appears to have the same location on the NA surface as in N9 NA. However previous attempts to observe this site by x-ray diffraction were unsuccessful. Here we report conditions for visualizing, by x-ray diffraction, Neu5Ac bound to the HB site of N9 NA and show that the sequence signature of side chains, which interact with this Neu5Ac, is largely conserved in all avian influenza viruses.

MATERIALS AND METHODS

Data Collection.

The NA enzyme was purified from influenza virus A/tern/Australia/G70C/75 virus as described (26) and crystallized by established procedures (21). The crystals were transferred to 20% glycerol while maintaining the concentration of the phosphate buffer before freezing in a cold stream of nitrogen gas at −166°C. X-ray diffraction data were collected on a R-axis IV Image Plate x-ray detector mounted on a MAC Science SRA M18XH1 rotating anode x-ray generator, operating at 47 kV and 60 mA with focusing mirrors. Two x-ray data sets were collected, namely wild-type NA complexed with Neu5Ac at 4°C (N9–4C) and 18°C (N9–18C). All crystals were soaked with 20 mM Neu5Ac for 4 hr and flash-frozen to −166°C before data collection. Listed on Table 1 are the data collection statistics for the two data sets.

Table 1.

X-ray data collection statistics for crystals of A/tern/Australia/G70C/75 N9 NA

Crystal N9-18C N9-4C*
Neu5Ac soaking temp.°C† 18 4
Total observations 164,615 291,377
Unique intensities 38,810 45,647
IP frames 80 60
Rmerge 8.8% 7.6%
〈Rsym〉‡ 8.9% 8.5%
Resolution 1.8 Å 1.7 Å
 Completeness total 83.6% 96.6%
 Last shell 68.2% 91.8%
 Range 1.92-1.8 Å 1.8-1.7 Å
〈I/σ〉 9.8 9.6
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*

N9-4C refined to R = 0.178 (Rfree = 0.205) with 369 waters added, for data between 6-1.7 Å with rmsd from ideal geometry of 0.016 Å and 1.90° for bonds and angles, and rms B value of 13.4. Eighty-two percent of residues were in the most favorable region on a Ramachandran Plot, with no outliers. 

†

20 mM for 4 hr, then flash-frozen to −166°C. 

‡

Averaged over the number of frames. 

Structure Refinement.

The refined structure at −166°C of wild type (27) was used as the reference structure. The location of the Neu5Ac moieties were obtained by examination of the difference Fourier of the complexed and the uncomplexed NA x-ray data using the phases of the uncomplexed refined atomic model with all active site water molecules removed. Both data sets revealed Neu5Ac in the active site of NA in a twisted-boat conformation, and active site water molecules as determined previously (14). The N9–4C complex showed the second Neu5Ac binding site clearly in the difference Fouriers. These moieties appeared as large positive features greater than 5 σ in the difference Fouriers, whereas for the N9–18C complex the difference Fouriers indicated only noise peaks (less than 3 σ) at the second site. The orientation of the Neu5Ac moiety in the chair conformation was unambiguous, and atomic models were built into these difference Fouriers.

The structure then was refined to 1.7 Å resolution for N9–4C using the refined wild-type structure and models for the Neu5Ac moieties at the two sites as a starting atomic model, using xplor (28). Water molecules in the active site and elsewhere were added by examining the difference Fourier peaks of observed and calculated structure factors of the atomic model that were greater than 5 σ. In the above xplor refinement all charges in charged amino acids were set to zero, as well as charged groups on the inhibitors. Crystallographic-based stereo chemical restraints (29) were used, and the structure retained good stereo geometry (see Table 1 for refinement statistics).

RESULTS

X-ray diffraction analysis of crystals of A/tern/Australia/G70C/75 N9 NA, soaked with 20 mM Neu5Ac soaked for 4 hr at 4°C, and subsequently flash-frozen to −166°C, reveals two Neu5Ac moieties bound to the NA. One is in the conserved active site in a twisted boat conformation as reported previously (14), the other is observed nearby about 14 Å away (the ketosidic oxygens of the two Neu5Ac moeties are about 21.3 Å apart), in the direction of Arg-371 and on the other side of the 367–372 loop (Figs. 1 and 2). This second site is in a chair conformation similar to what is found in the Neu5Ac binding sites of HA (30). A similar analysis with crystals soaked with 20 mM Neu5Ac soaked for 4 hr at 18°C and then flash-frozen to −166°C indicates that this second Neu5Ac binding site is unoccupied.

Figure 1.

Figure 1

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A molecular surface rendered image (42) of a tetrameric head of A/tern/Australia/G70C/75 N9 NA viewed from above the molecule. The active site residues interacting with the Neu5Ac moiety (twist-boat conformation) in the catalytic sites are show in green. The residues that interact with the Neu5Ac moiety (chair conformation) in the HB sites are colored yellow (conserved in all avian strains) and blue (conserved in N9 stains). (Inset) A magnified region (×1.6) in the vicinity of these two sites of one subunit, illustrating the deep pocket of the catalytic site and the flat surface of the HB site of the enzyme.

Figure 2.

Figure 2

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(A) A stereo drawing (43) of the refined x-ray atomic model of the Neu5Ac (orange) bound in the HA site of A/tern/Australia/G70C/75 NA with Neu5Ac soaked at 4°C, showing all the amino acids (green), and water molecules (red) making contact with the moiety. Atomic interactions are shown in broken lines. Oxygen, nitrogen, and carbon atoms are colored red, blue, and black, respectively. The protein backbone is represented by a yellow tube. (B) The two-Fo-Fc electron density map (blue caged-mesh contour at 1.6 σ level) of the Neu5Ac in the same orientation, using the refined phases of the complex, where Fo and Fc are the observed and calculated structure factors, respectively.

The location of this second site in the N9–4C complex (Fig. 2) is precisely where it was predicted by site-directed mutagenesis (22) in wild-type A/tern/Australia/G70C/75 N9 NA and in A/FPV/Rostock/34 N1 NA (25). One of the oxygens of the carboxylate group of Neu5Ac has a hydrogen bond interaction with the hydroxyl oxygen of Ser-367 and the other carboxylate oxygen hydrogen bonds to the amide on the side chain of Asn-400. The main chain carbonyl oxygen of Asn-400 interacts with both the 4-hydroxyl oxygen and the nitrogen of the 5-acetamido group of Neu5Ac. This nitrogen also interacts with the hydroxyl oxygen of Ser-372. The methyl carbon of the 5-acetamido group has a hydrophobic interaction with Trp-403, sitting 3.5 Å above the six-membered ring of the tryptophan similar to an interaction in the HA Neu5Ac binding site (31) with the plane of the acetamido group lying almost parallel to the plane of the tryptophan rings. The distal hydroxyl oxygen (O9) of the 6-triol group of Neu5Ac interacts with the ɛ amino of Lys-432, the next hydroxyl (O8) interacts with the hydroxyl oxygen of Ser-370, and the proximal hydroxyl oxygen is exposed to the solvent.

Thus three loops are on NA that primarily are responsible for the formation of this HB site. The first loop containing residues 367-SIASRS-372 is involved in three interactions with Neu5Ac through the three serine residues 367, 370, and 372. A second loop containing residues 400-NTSW-403 is involved with three interactions with Neu5Ac through Asn-400 side chain and main chain carbonyl oxygen, and a hydrophobic interaction with Trp-403. A third loop containing Lys-432 interacts with the distal hydroxyl of the 6-triol of Neu5Ac via the ɛ-amino nitrogen of the side chain.

A water molecule (Fig. 2) was found hydrogen-bonded to both carboxylate oxygens of Neu5Ac, and another to both the proximal hydroxyl oxygen of the 6-triol group of Neu5Ac and to the ring oxygen. A third water molecule interacts with the carbonyl oxygen of the 5-acetamido group of Neu5Ac, and another with the ketosidic oxygen of Neu5Ac.

DISCUSSION

We have shown that six residues on three separate loops of N9 NA interact directly with the Neu5Ac in the second Neu5Ac binding site. These are the three serine residues (367, 370, and 372) in the loop containing residues 367-SIASRS-372, Asn-400 and Trp-403 in the loop containing 400-NTSW-403, and Lys-432 in a third loop. Not all of these six residues are conserved in the NAs, which have been shown to possess the HB activity (22, 23, 25). Thus the Lys-432 interaction is not absolutely necessary for binding, as a site-directed mutant of A/Tokyo/3/67 N2 NA gained the HB site activity with a glutamine at this position (23). It was shown (22) that antigenic variants with amino acid substitutions at 432 had a minimal effect on HA activity on N9. Furthermore A/FPV/Rostock/34 N1NA has HB activity (25) with an asparagine at position 432, and an isoleucine at position 400. This would indicate that the interactions with the distal hydroxyl (O9) of the 6-triol group (which interacts with Lys-432 in N9 NA) could interact with the protein via a water molecule as in the active site of bacterial sialidases (32) or some as-yet-unidentified residue on the A/FPV/Rostock/34 N1 NA and A/Tokyo/3/67 N2 NA. Similarly the carboxylate oxygen of Neu5Ac that interacts with Asn-400 in N9 NA would have to interact with A/FPV/Rostock/34 NA in a different manner.

In summary, the sequence variation at residues 400 and 432 in NAs with known HB activity indicates that these residues are not essential for HB activity, and the triple serine SxxSxS loop of 367 to 372 and Trp-403 is a minimal signature for HB binding in NA.

This is consistent with the observation that mutations (22) at residues 367, 370, and 372 markedly reduced or abolished HB activity and that an A369D mutation would interfere with the HB site in N9 NA. Also the insensitivity of the mutation I368R to HB activity arises because the side chain at 368 points away from the HB site (see Fig. 2). It therefore would require a L370S and a R403W mutation as a minimum requirement for transfer of HB activity (23) to A/Tokyo/3/67 NA.

Sequence comparisons of the different subtypes of NA in the region of these three loops indicate that only the N9 subtype has all six contact residues as defined here. It should be noted that Gly-373, which is involved in a turn between two β-strands on the fifth β-sheet positions the invariant catalytic residue Arg-371, and Ser-404, which positions Trp-403 in the HB site, both are conserved in all type A NAs. However, if a comparison is made using the triple serine and Trp-403 signature (Table 2), all known strains from N1 to N9, which carry the signature, are avian (or equine), with the exception of the two human N2 strains RI5+/57 and Leningrad/134/57. Furthermore, all avian sequences carry this signature, and in general human and swine NAs do not. We propose that the signature has a functional relevance to avian influenza.

Table 2.

A comparison of sequences of influenza virus isolates (* avian strains) that contain the HB signature (large bold)

366–373 399–404 430–433 Influenza strain Subtype
KSTSSRSG AITDWS RPNH A/FPV/Rostock/34 N1*
KSTSSRSG AITDWS RPKE A/Parrot/Ulster/73 N1*
ISKESRSG DNNNWS RPQE A/RI/5+/57 N2
ISKDSRSG DNNNWS RPQE A/Leningrad/134/57 N2
ISKDSRSG DNNNWS RPQE A/duck/HongKong/24/76 N2*
ISKDSRSG DNNNWS RPQE A/Chicken/Pennsylvania/1370/83 N2*
ISVSSRSG NNKNWS RKQE A/Duck/Alberta/60/76 N5*
ISVSSRSG NNKNWS RKQE A/Shearwater/72 N5*
ISPRSRSG DNSNWS RPEE A/FPV/Weybridge N7*
ISRTSRSG DNLNWS RKQE A/Equine/Miami/1/63 N8
ISRTSRSG DNLNWS RKQE A/Duck/Ukraine/1/63 N8*
ISRTSRSG DNLNWS RKQE A/Quail/Italy/1117/65 N8*
ISRTSRSG DNLNWS RKQE A/Equine/SaoPaulo/6/69 N8
ISRTSRSG DNLNWS RKQE A/Equine/Algiers/72 N8
ISRTSRSG DNLNWS RKQE A/Duck/Chabarovsk/1610.72 N8*
ISRTSRSG DNLNWS RKQE A/Duck/Memphis/928/74 N8*
ISRTSRSG DNLNWS RKQE A/Turkey/Minnesota/501/78 N8*
ISRTSRSG DNLNWS RKQE A/Duck/Hokkaido/8/80 N8*
ISRTSRSG DNLNWS RKQE A/GuineaFowl/NY/4-3587/84 N8*
ISRTSRSG DNLNWS RKQE A/Duck/Burjatia/652/88 N8*
ISRTSRSG DNLNWS RKQE A/Herring Gull/DE/677/88 N8*
ISRTSRSG DNLNWS RKQE A/Equine/Jilin/1/89 N8
ISRTSRSG DNLNWS RKQE A/Mullard/Edmonton/220/90 N8*
ISRTSRSG DNLNWS RKQE A/Equine/Alaska/1/91 N8
ISIASRSG LNTDWS RPKE A/tern/Australia/G70C/75 N9*
ISIASRSG LNTDWS RPKE A/whale/Maine/1/84 N9
ISTASRSG LNTDWS RPKE A/RuddyTurnstone/NJ/60/85 N9*
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   Only the three loops that interact with the Neu5Ac HB site of G70C N9 are listed. In bold are residues in contact with Neu5Ac in N9. The sequence data are from the GenBank database. 

It has been shown (24) that the HB site on A/tern/Australia/G70C/75 NA binds a moiety on red cells that is not cleavable by A/tern/Australia/G70C/75 NA, a consequence of the moiety either being Neu5Ac in a noncleavable linkage or something other than Neu5Ac. The biological significance of this HB site has not been determined, but the above results would indicate that it may function as a lectin to some avian cell receptor, which is unaffected by influenza NA activity. Influenza is normally an asymptomatic infection in birds (33), but replicates preferentially in the cells lining the intestinal tract of waterfowl (34, 35), where all of the different subtypes of influenza A have been isolated. This has led to the proposition that waterfowl are the primary vectors for the global spread of the disease (36) through fecal droppings. This avirulent adaptation of the virus to avian species enables it to survive and persist in a vast global reservoir.

If this HB signature relates to some as-yet-unidentified role in avian influenza, then it could be inferred that the first human 1957 pandemic N2 strains carrying the HB signature (RI5+/57 and Leningrad/134/57) most probably were derived from a genetic reassortment event with an avian strain (37). This HB signature since has disappeared under antigenic drift in post-1957 human and swine stains, indicating that it serves no biological function in pathogenesis of influenza in humans and pigs.

One of the puzzling aspects of this study is the relationship of this site to equine strains. Equine strains, which appear across all subtypes, almost always carry this HB signature, and it is possible they share with avian species similar carbohydrates that are the natural ligands for this HB site. Furthermore, the signature is present in the RI5+/57 viral strain, which had high affinity to normal horse serum (38), but is absent in the RI5-/57 strain, which had a low affinity to normal horse serum. The RI5+/57 strain was selected as a result of serial passaging in chicken embryo. Although circumstantial evidence indicated that the difference between the RI5+/57 and RI5-/57 strains probably was due to different affinity of the HA for the inhibitor in horse serum (38–40), it would appear that the differences in the NA at the HB site could be an important factor in the different properties of the two strains. When horse serum was treated with Vibrio cholera extract, RI5+/57 virus retained binding to horse serum, whereas RI5-/57 virus did not do so, further indicating that either the HA was different from most HAs that don’t bind to V. cholera-treated serum, or that this HB site on the NA was responsible for this difference.

Sequences of the paramyxovirus HN protein, which carries both NA and HA function, were examined based on a sequence alignment that is consistent with the viral NA fold (41). This HB signature was not able to be identified.

Acknowledgments

We thank Pat Pilling for technical assistance and Colin Ward and Brian Smith for helpful discussions and reading this manuscript.

Footnotes

This paper was submitted directly (Track II) to the Proceedings Office.

Abbreviations: NA, neuraminidase; Neu5Ac, sialic acid; HA, hemagglutinin; HB, hemabsorbing.

Data deposition: The atomic coordinates reported in this paper have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY 11973 (reference no. 1MWE for the A/tern/Australia/G70C/75 NA/Neu5Ac complexed at 4°C).

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