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The Journal of Biological Chemistry logoLink to The Journal of Biological Chemistry
. 2012 Jul 30;287(41):34547–34557. doi: 10.1074/jbc.M112.357061

Theoretical Investigation on the Binding Specificity of Sialyldisaccharides with Hemagglutinins of Influenza A Virus by Molecular Dynamics Simulations

Thanu R K Priyadarzini ‡,1, Jeyasigamani F A Selvin ‡,2, M Michael Gromiha §, Kazuhiko Fukui ¶,3, Kasinadar Veluraja ‡,3,4
PMCID: PMC3464561  PMID: 22846994

Background: Recognition of terminal sialyldisaccharides by influenza A hemagglutinin initiates the infection process of influenza.

Results: MD simulations on sialyldisaccharide-hemagglutinin (H1, H3, H5, and H9) complexes reveal the molecular basis of specific recognition.

Conclusion: The order of the binding specificity of Neu5Acα(2–3)Gal and Neu5Acα(2–6)Gal is H3 > H5 > H9 > H1 and H1 > H3 > H5 > H9, respectively.

Significance: The insights from this study will help in designing carbohydrate-based therapeutics against influenza viral infections.

Keywords: Biophysics, Carbohydrate-binding Protein, Carbohydrate Structure, Molecular Biology, Molecular Dynamics, Molecular Modeling, Viral Protein

Abstract

Recognition of cell-surface sialyldisaccharides by influenza A hemagglutinin (HA) triggers the infection process of influenza. The changes in glycosidic torsional linkage and the receptor conformations may alter the binding specificity of HAs to the sialylglycans. In this study, 10-ns molecular dynamics simulations were carried out to examine the structural and dynamic behavior of the HAs bound with sialyldisaccharides Neu5Acα(2–3)Gal (N23G) and Neu5Acα(2–6)Gal (N26G). The analysis of the glycosidic torsional angles and the pair interaction energy between the receptor and the interacting residues of the binding site reveal that N23G has two binding modes for H1 and H5 and a single binding mode for H3 and H9. For N26G, H1 and H3 has two binding modes, and H5 and H9 has a single binding mode. The direct and water-mediated hydrogen bonding interactions between the receptors and HAs play dominant roles in the structural stabilization of the complexes. It is concluded from pair interaction energy and Molecular Mechanic-Poisson-Boltzmann Surface Area calculations that N26G is a better receptor for H1 when compared with N23G. N23G is a better receptor for H5 when compared with N26G. However, H3 and H9 can recognize N23G and N26G in equal binding specificity due to the marginal energy difference (≈2.5 kcal/mol). The order of binding specificity of N23G is H3 > H5 > H9 > H1 and N26G is H1 > H3 > H5 > H9, respectively. The proposed conformational models will be helpful in designing inhibitors for influenza virus.

Introduction

Influenza is a zoonotic disease caused by RNA viruses of the Orthomyxoviridae family. Influenza viruses are roughly spherical in shape with glycoprotein spikes on their cell surface, and their genome consists of eight RNA fragments that encode 10 proteins. Among the surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA)5 are critical for the biology of influenza viruses. Based on the serological differences, influenza viruses are divided into three types: A, B, and C (1–7). Of the three types, influenza A viruses can infect a variety of animals, including poultry, human, pigs, horse, murine mammals, and carnivore animals (8–10). The spikes of HA bind to sialic acids that are found on the surface of the host cell membrane and mediate virus attachment to target cells (11–15). NA cleaves the α-glycosidic linkage between the terminal sialic acid and the penultimate sugar residue, facilitating elution of virus progeny from infected cells and preventing self-aggregation of the virus. Depending on the type of glycoproteins, HA and NA, influenza A viruses are further classified into different subtypes. So far, 16 different subtypes of HA and 9 subtypes of NA have been found. Of the 16 subtypes of HA, most are isolated from avian species and a few from equine (H3 and H7), seals (H3, H4 and H7), whales (H1 and H13), and swine (H1, H3 and H9) (16–22). Influenza A subtypes H1, H2, and H3 had caused five pandemics in last century, viz. Spanish 1918 (H1), Asian 1957 (H2), Hong Kong 1968 (H3), Russian 1971 (H1), and Mexican flu 2009 (H1) (23–26). The receptor binding specificity of influenza viruses is related to the recognition of cellular sialic acids by the viral hemagglutinin (27–30).

Sialic acids are significant sugar molecules typically found at the outermost ends of N-glycans, O-glycans, or glycosphingolipids. They exist in diversified forms that arise from the modifications of 5th position in neuraminic acid and additional substitutions at the hydroxyl group on the 4th, 7th, 8th, and 9th carbon, and the other diversified forms are generated by the linkages from the 2nd carbon to the penultimate sugar. The most common linkages are Neu5Acα(2–3)Gal (N23G) and Neu5Acα(2–6)Gal (N26G). These structural diversities of sialic acid can govern the recognition by a variety of sialic acid-binding lectins such as influenza virus hemagglutinin. The receptor specificity of influenza viruses is defined in terms of their recognition of sialic acid species (Neu5Ac, Neu5Gc, Neu4,5Ac2, etc.), and the type of glycosidic linkage between sialic acid and the penultimate galactose (31–33). In general, influenza A and influenza B viruses recognize Neu5Ac/Neu5Gc, and influenza C virus binds to 9-O-acetylated sialic acid (34–37). Influenza A viruses isolated from human and avian recognize N26G and N23G, respectively. Swine influenza virus can bind with both N23G and N26G.

A single amino acid change in the receptor-binding site of hemagglutinin of influenza A virus alters the binding specificity from N23G to N26G (38, 39). It has been reported by Taubenberger and co-workers (40) that mutation at positions 190 and 225 (E190D and G225D) and 226 and 228 (Q226K and G228S) will affect the binding specificity to sialic acid.

Molecular dynamics (MD) simulations are used to study the conformational behaviors of carbohydrates and their binding with proteins (41–45). In this study, 10-ns MD simulations are carried out for the complexes of H1-N23G, H1-N26G, H3-N23G, H3-N26G, H5-N23G, H5-N26G, H9-N23G, and H9-N26G to understand the binding specificity of sialyldisaccharides with hemagglutinin H1, H3, H5, and H9 of influenza A virus, and the probable binding models are proposed for the above complexes. The pair interaction energies of the complexes are calculated using Nanoscale Molecular Dynamics (NAMD). Hydrogen bonding patterns, which stabilize the complexes, are found, and the binding free energy due to the water molecule mediating the interaction is also calculated using NAMD for each water-mediated hydrogen-bonding interaction. The binding free energy of the complexes is calculated using MM-PBSA calculation.

COMPUTATIONAL DETAILS

The molecular structures of N23G and N26G along with the glycosidic torsional angles Φ and Ψ are shown in Fig. 1, A and B. The geometry of N23G and N26G is generated using the standard bond length, bond angles, and torsional angles (46, 47). The three-dimensional coordinates of influenza A hemagglutinins were taken from the Protein Data Bank of Research Collaboratory for Structural Bioinformatics. In this study, we have used the HAs of influenza A strains H1, H3, H5, and H9 and the respective Protein Data Bank codes are 1RV0, 1MQM, 1JSN, and 1JSH (48–50). The number of amino acids in H1, H3, H5, and H9 are 324, 318, 321, and 317 respectively. N23G and N26G are modeled into the binding site of H1, H3, H5, and H9. The force field parameters gaff (51) and ff99 (52) are used to prepare the input parameters for sugar and protein, respectively. Care is taken to neutralize the entire protein-sugar complex. The total system is solvated with TIP3P water molecules using solvents library of AMBER9. The temperature of the ensemble is kept at 300 K, and constant pressure is maintained. The cutoff for the nonbonding interactions was set to 10 Å. We have used sander of AMBER9 (53) to perform the MD simulation. MD simulations of 10-ns duration was carried out for the complexes H1-N23G, H1-N26G, H3-N23G, H3-N26G, H5-N23G, H5-N26G, H9-N23G, and H9-N26G. The trajectories are collected over every picosecond, and a total of 10,000 structures are collected for each simulation. To get insights on the atomistic level interactions between the receptor sugar residue and HA protein, graphical software VMD (54) is used along with the FORTRAN programs developed in-house. The interaction energy between the interacting amino acid residues and the receptor sugar residue is computed by NAMD (55). The binding free energy between the protein and the receptor is calculated using MM-PBSA module of AmberTools. MOLSCRIPT (56) is used to render the molecular interactions.

FIGURE 1.

FIGURE 1.

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Schematic representation. A, Neu5Acα(2–3)Gal (ΦN23G = C1-C2-O2-C3; ΨN23G = C2-O2-C3-H3) (N23G). B, Neu5Acα(2–6)Gal (N26G) (ΦN26G = C1-C2-O2-C6; ΨN26G = C2-O2-C6-H61; ωN26G = O2-C6-C5-O5 (H61 is the hydrogen that makes an angle of H61-C6-C5-O5 = 120° when ωN26G = 0°); χn = C5-N5-C10-C11; χ1 = C5-C6-C7-C8; χ2 = C6-C7-C8-C9; χ3 = C7-C8-C9-O9; χ4 = C1-O5-C5-C6).

RESULTS

The results of 10-ns MD simulations carried out for eight protein-carbohydrate complexes, namely H1-N23G, H1-N26G, H3-N23G, H3-N26G, H5-N23G, H5-N26G, H9-N23G, and H9-N26G, were analyzed using VMD and in-house developed FORTRAN programs. Information about conformational flexibility of the receptors (N23G and N26G), atomistic level interaction between functional groups of receptor (carboxylate, acetyl, glycerol side chain, hydroxyl, and hydroxymethyl), and the binding site residues were obtained to gain insights on the binding affinity of the receptors with different hemagglutinins of influenza A virus. The possible conformations of sialyldisaccharides were deduced from the glycosidic torsional map, and the associated interaction energies of the HA-sialyldisaccharide complexes were calculated. The interaction patterns were proposed based on rigorous analysis of the MD simulation trajectories and superimposed trajectories of various frames having similar conformation for sialyldisaccharides. The relative binding free energies of the sialyldisaccharides-protein complexes were calculated using MM-PBSA module of AmberTools. The binding free energy of the water molecule, which is involved in water mediation, was calculated using NAMD. The conformations of the receptors N23G and N26G at the binding site of hemagglutinin were dictated by glycosidic conformational torsional angles denoted as Φ and Ψ. In addition to the glycosidic torsional angles, N26G possesses an exocyclic torsional angle (ω). The energy of the complexes of each hemagglutinin with N23G and N26G can be compared with each other because the number of atoms in each simulation of similar hemagglutinin was the same.

Conformation of Sialyldisaccharides N23G and N26G at the Binding Pocket of HAs

The conformational flexibilities of the receptors N23G and N26G were examined in terms of acetamido group conformation (χn = C5-N5-C10-C11), glycerol side chain conformation (χ1 = C5-C6-C7-C8; χ2 = C6-C7-C8-C9; and χ3 = C7-C8-C9-O9) of sialic acid, and the hydroxymethyl group conformation (χ4 = C4-C5-C6-O6) of galactose. The 10-ns MD simulations on the complexes indicated that in all the HA-N23G complexes χn preferred the trans conformation; χ1 preferred trans for the complexes H1-N23G, H5-N23G, and H9-N23G. For H3-N23G complex, χ1 preferred trans and g−. χ2 preferred g+ for H1-N23G, H5-N23G, and H9-N23G complexes and trans for H3-N23G complex. The terminal hydroxyl group conformation χ3 preferred trans conformation for H1-N23G and H3-N23G complexes, whereas both trans and g+ conformations are preferred for H5-N23G and H9-N23G complexes. χ4 of galactose preferred g+ in all the complexes.

In HA-N26G complexes, χn and χ1 preferred trans conformation in all the complexes. χ2 preferred trans conformation for H1-N26G and H3-N26G and both trans and g+ conformations for H5-N26G and H9-N26G complexes. In all the complexes χ3 preferred trans and g+ conformations.

The overall structural flexibility of the HA-sialyldisaccharide complexes was measured in terms of root mean square deviation of backbone, and the root mean square deviation trajectories are given as supplemental Fig. S1.

N23G and N26G at the Binding Pocket of H1, Conformation, and Interaction Energy

The conformational features of the receptor N23G and N26G in the binding site of H1 hemagglutinin were computed, and the conformer distributions based on glycosidic torsions are given in Fig. 2, A and B, respectively. It is evident from Fig. 2A that N23G exists in three different conformational regions inside the binding pocket of H1, and the glycosidic torsional values (ΦN23G-H1 and ΨN23G-H1) of these regions are AN23G-H1 (−150 and −30°), BN23G-H1 (−100 and −50°), and CN23G-H1 (−70 and 0°) with respective population propensities of 37, 35, and 16%. The three regions are denoted as BM1, BM2, and BM3, respectively. When N23G exists in binding mode 1 (BM1), the carboxylate group formed hydrogen bonds with the hydroxyl of Thr-133, backbone amide group of Ala-134, and side chain nitrogen of Gln-223. Also, the carboxylate group formed a water-mediated hydrogen bond with side chain hydroxyl of Ser-142. O4 hydroxyl of sialic acid interacted with Arg-130 through water mediation. Amide group of sialic acid was hydrogen-bonded with the backbone carbonyl of Val-132. O7 hydroxyl of sialic acid bonded with the hydroxyl group of Tyr-91. The glycerol side chain hydroxyl oxygens O8 and O9 formed either a direct or water-mediated hydrogen bond with the side chain oxygen of Asp-187. The hydroxymethyl group of galactose residue made a hydrogen bond with the backbone amide group of Ala-224. In BM1, a total of 10 hydrogen bonds (six direct and four water-mediated) are involved in the structural stabilization of the receptor at the binding site of H1 (Fig. 3A), and the interactions are given in Table 1. In binding mode 2 (BM2), the following interactions were observed between the receptor and protein. The interactions between the binding site residues and the carboxylate group, O8 and O9 hydroxyl oxygens of glycerol side chain, were similar to those of BM1. However, the hydroxymethyl group of galactose made a hydrogen bond with the backbone oxygen of Gly-222 instead of Ala-224. A total of nine hydrogen bonds (five direct and four water-mediated) are involved in the structural stabilization of H1-N23G in BM2 (Fig. 3B). At the CN23G-H1 region (BM3), the interaction between binding site residues and sialic acid was similar as was that of BM2. However, the second residue galactose did not make any hydrogen bonds with the binding site residues. Interaction energy between the receptor and the binding site residues of H1 (Tyr-91, Arg-130, Val-132, Thr-133, Ala-134, His-180, Asp-187, Gln-223, Ala-224, and Gly-225) was calculated using NAMD and plotted with respect to time as shown in Fig. 4A. It is evident from Fig. 4A that interaction energy falls into three distinct regions, and this substantiates the above binding modes. In the interaction energy plot, the energy was minimum for 0–3500 ps and corresponded to BM1. The second dominant region was mainly observed between 5000 and 7500 ps, and it corresponded to BM2. The interaction energy graph clearly confirmed that although H1 can accommodate N23G in three distinct modes, BM1 was the best binding mode for H1.

FIGURE 2.

FIGURE 2.

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Glycosidic torsional map for H1-N23G (A), H1-N26G (B), H3-N23G (C), H3-N26G (D), H5-N23G (E), H5-N26G (F), H9-N23G (G), and H9-N26G (H).

FIGURE 3.

FIGURE 3.

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Interactions between H1 and receptor H1-N23G at BM1 (A), H1-N23G at BM2 (B), H1-N26G at BM1 (C), and H1-N26G at BM2 (D).

TABLE 1.

Interactions between receptors (N23G and N26G) and the active site residues of H1, H3, H5, and H9 hemagglutinins

Receptor Binding modes Receptor atom Protein atoms in H1 Binding free energy due to water mediation Protein atoms in H3 Binding free energy of due to water mediation Protein atoms in H5 Binding free energy due to water mediation Protein atoms in H9 Binding free energy due to water mediation
kcal/mol kcal/mol kcal/mol kcal/mol
N23G Binding mode1 (BM1) O1 Thr-133 OG1 − Ser-128 OG − Ser-132 OG − Ser-130 OG −
Ala-134 N − Ser-129 N − Ser-133 N − Ser-131 N
Gln-223 NE2 − Ser-129 OG − Ser-133 OG − Ser-131 OG
WAT-Ser-142 OG −3.3 ± 0.3 Asn-137 ND2 − WAT-Ser-141 OG −3.4 ± 0.3
Gln-218 NE2 −
O4 WAT-Arg-130 O −3.2 ± 0.4 − − WAT-Ser-129 O −3.1 ± 0.7 WAT-Thr-129 O −3.1 ± 0.5
N5 Val-132 O − Gly-127 O − Val-131 O − Thr-129 O −
O7 Tyr-91 OH − − − WAT-Glu-186 OE2 −3.5 ± 0.5 − −
O8 WAT-Asp-187 OD1 −3.4 ± 0.6 Gln-218 NE2 − Glu-186 OE1 − Tyr-91 OH −
O9 WAT-Asp-187 OD1 −3.4 ± 0.6 Tyr-90 OH − Tyr-91 OH − Tyr-91 OH −
Glu-182 OE2 − His-179 NE2 − Asn-173 ND2
His-175 NE2 − Asn-182 ND2 −
O1B − − − − − − − −
O2B − − − − WAT-Lys-189 NZ −3.4 ± 0.3 − −
O3B − − − − − − − −
O4B − − Ser-129 OG − − − − −
O6B Ala-224 N − Gly-217 O − Asn-182 ND2 − Gly-215 O −
WAT-Lys-218 NZ −3.1 ± 0.2
Binding mode2 (BM2) O1 Thr-133 OG1 − − − Ser-132 OG − − −
Ala-134 N − Ser-133 N −
Gln-223 NE2 − Ser-133 OG −
WAT-Ser-142 OG −3.3 ± 0.3 WAT-Ser-141 OG −3.4 ± 0.3
O4 WAT-Arg-130 O −3.2 ± 0.4 − − WAT-Ser-129 O −3.1 ± 0.7 − −
N5 Val-132 O − − − Val-131 O − − −
O7 − − − − Glu-186 OE2 − − −
O8 WAT-Asp-187 OD1 −3.4 ± 0.6 − − Gln-222 NE2 − − −
Tyr-91 OH −
O9 WAT-Asp-187 OD1 −3.4 ± 0.6 − − His-179 NE2 − − −
Glu-186 OE1 −
O2B − − − − − − − −
O3B − − − − − − − −
O4B − − − − − − −
O6B Gly-222 O − − − − − − −
N26G Binding mode1 (BM1) O1 Thr-133 OG1 − Ser-128 OG − Ser-132 OG − −
Ala-134 N − Ser-129 N − Ser-133 N − Ser-130 OG
WAT-Ser-142 OG −3.3 ± 0.5 Ser-129 OG − Ser-133 OG − Ser-131 OG
Gln-223 NE2 − Asn-137 ND2 − WAT-Ser-141 OG −3.5 ± 0.4 Ser-131 N
O4 WAT-Arg-130 O −3.1 ± 0.3 − − WAT-Ser-129 O −3.2 ± 0.2 − −
N5 Val-132 O − Gly-127 O − Val-131 O − Thr-129 O −
O7 − − − − WAT-Lys-189 NZ −3.4 ± 0.6 − −
O8 Tyr-91 OH − Gln-218 OE1 − − − Tyr-91 OH −
O9 His-180 NE2 − Tyr-90 OH − Tyr-91 OH − − −
WAT-Gln-223 OE1 −3.6 ± 0.8 His-175 NE2 − Asn-182 ND2 −
WAT-Ala-224 N −3.6 ± 0.8 Glu-186 OE2
WAT-Gly-225 N −3.6 ± 0.8
O2B WAT-Lys-219 NZ −3.1 ± 0.4 − − − − − −
O3B Gly-222 O − Gly-217 O − WAT-Lys-218 NZ −3.2 ± 0.3 Gly-215 O −
O4B Gly-222 O − Gly-217 O − − − Gly-215 O −
O6B − − − − − − − −
Binding mode2 (BM2) O1 Thr-133 OG1 − Ser-128 OG − − −
Ala-134 N − Ser-129 N −
Gln-223 NE2 − Ser-129 OG −
WAT-Ser-142 OG −3.3 ± 0.5 Asn-137 ND2 −
O4 WAT-Arg-130 O −3.1 ± 0.3 − − − − −
N5 Val-132 O − Gly-127 O − − − −
O7 − − − − − − −
O8 Tyr-91 OH − Gln-218 OE1 − − − −
O9 His-180 NE2 − Tyr-90 OH − − − −
WAT-Gln-223 OE1 −3.6 ± 0.8 His-175 NE2 −
WAT-Ala-224 N −3.6 ± 0.8
WAT-Gly-225 N −3.6 ± 0.8
O2B − − Gly-217 O − − − −
O3B − − Gly-217 O − − − −
O4B − − − − − − −
O6B − − − − − − −
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FIGURE 4.

FIGURE 4.

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Interaction energy for the complexes H1-N23G (A), H1-N26G (B), H3-N23G (C), H3-N26G (D), H5-N23G (E), H5-N26G (F), H9-N23G (G), and H9-N26G (H).

In the MD trajectories of the H1-N26G complex, the disaccharide N26G preferred two distinct conformations AN26G-H1 and BN26G-H1 with the glycosidic torsional angle (ΦN26G-H1 and ΨN26G-H1) values of −150 and 70° and −70 and 70° with the population propensities of 35 and 60%, respectively, and the exocyclic torsional angle predominantly preferred 70°. In the BN26G-H1 (BM1) region, N26G was buried well within the binding site and made a cluster of direct and water-mediated hydrogen bonds with the binding site residues. The interactions of sialic acid with the binding site were as follows: carboxylate group made hydrogen bonds with a side chain hydroxyl of Thr-133, backbone nitrogen of Ala-134, and a side chain of Gln-223; through water mediation with a hydroxyl of Ser-142; O4 hydroxyl of sialic acid with a carbonyl of Arg-130 through water mediation; acetamido group forms hydrogen bond with carbonyl of Val-132; O8 hydroxyl with polar side chain of Tyr-91; O9 hydroxyl with side chain nitrogen of His-180; O9 through water mediation with a side chain hydroxyl of Gln-223, amide nitrogen of Ala-224 and carbonyl of Gly-225. Interactions of galactose with the binding site residues are given below: O3 and O4 hydroxyl with carbonyl of Gly-222; O3/O2 hydroxyl with Lys-219 through water mediation. These interactions are given in Table 1 (Fig. 3C). A total of 14 hydrogen bonds (eight direct and six water-mediated) stabilize the complex in this binding mode (BM1). When the N26G prefers AN26G-H1 (BM2), the interaction between sialic acid and the binding site residues are similar to BM1. However, the galactose residue does not interact with the binding site residues. A total of 11 hydrogen bonds (six direct and five water-mediated) are involved in the structural stabilization of this complex in BM2 (Fig. 3D). These binding modes are reflected in interaction energy plot of H1-N26G (Fig. 4B), which exhibits two distinct modes. The NAMD-calculated interaction energy difference between H1-N23G and H1-N26G was 19.0 ± 0.28 kcal/mol, with H1-N26G being the minimum. The binding free energy of the complex H1-N23G and H1-N26G was calculated by using MM-PBSA calculation and the results were that the relative binding free energy difference between the two complexes was 6.4 ± 5.6 kcal/mol, with H1-N26G is being minimum. The atomistic level of interaction between the binding site residues and the receptor, the interaction energy analysis of the complexes, and the binding energy between the complexes show that N26G is the better receptor for H1 when compared with N23G.

N23G and N26G at the Binding Pocket of H3, Conformation and Interaction Energy

The conformational flexibility of the receptor N23G in the complex H3 is shown in Fig. 2C, and it shows that a single glycosidic torsional region (ΦN23-H3 and ΨN23-H3) is preferred and is denoted as AN23G-H3. The preferred glycosidic torsional angle was −90 and −50°. The atomistic level interactions between the receptor and the binding site residues were analyzed and are given in Table 1. A cluster of 12 direct hydrogen bonds were involved in stabilizing the H3-N23G complex. The flexibility of the receptor N23G was limited to a single rigid conformation, and this was due to the interactions of both the residues of N23G with H3 (Fig. 5A). The interaction energy between interacting amino acid residues (Tyr-90, Gly-127, Ser-128, Ser-129, Asn-137, His-175, Glu-182, Gly-217, and Gln-218) of H3 and N23G has been computed, and a single binding mode is reflected (Fig. 4C).

FIGURE 5.

FIGURE 5.

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Interactions between H3 and receptor H3-N23G at BM1 (A), H3-N26G at BM1 (B), and H3-N26G at BM2 (C).

In the H3-N26G complex, the receptor has two modes of binding via BM1 (AN26G-H3) with the glycosidic torsion of −70 and 50° and BM2 (BN26G-H3) with −90 and −50° as given in Fig. 2D. The respective population propensities for the two binding modes are 42 and 31%. The exocyclic torsional value is 70°. The atomistic level interaction between the receptor N26G and the binding site residues in BM1 are given in Table 1. A total of 10 hydrogen bonds are involved in the structural stabilization of the complex H3-N26G at BM1 as shown in Fig. 5B. In BM2, both the residues were interacting with the binding site residues of H3. In BM1, the O2 and O3 hydroxyl of galactose interacts with H3 and in BM2, and the O3 and O4 hydroxyl of galactose interacts with H3 (Table 1), and this is reflected in the interaction energy plot (Fig. 4D). The atomistic level interaction in BM2 is shown in Fig. 5C. The difference in interaction energy between H3-N23G and H3-N26G was calculated, and it suggested that H3-N26G has 2.1 ± 0.32 kcal/mol higher energy than H3-N23G. This energy difference can be attributed to the differences in the number of hydrogen bonds formed between the receptor and protein complex. The relative binding free energy of the complexes was calculated using the MM-PBSA module and was 3.0 ± 4.7 kcal/mol with H1-N23G being the minimum. Based on the atomistic level interactions between the receptor and the binding site residues of H3, interaction energy analysis, and the binding free energy of the complexes, it was concluded that N23G is the better receptor for H3 when compared with N26G.

N23G and N26G at the Binding Pocket of H5, Conformation and Interaction Energy

The analysis on the complex dynamics of H5-N23G indicates that H5 can accommodate N23G in two different modes denoted as regions AN23G-H5 (BM1) and BN23G-H5 (BM2), and the respective preferred glycosidic torsional angles for ΦN23G-H5 and ΨN23G-H5 are −120 and −50° and −70 and 0° (Fig. 2E). In BM1, sialic acid makes eight direct hydrogen bonds and three water-mediated hydrogen bonds and galactose makes one direct hydrogen bond and two water-mediated hydrogen bonds with the binding site residues (Table 1). A total of 14 hydrogen bonds (nine direct and five water-mediated) are involved in stabilizing the complex in BM1 (Fig. 6A). In BM2, sialic acid interactions are similar to BM1 but the hydroxymethyl group of galactose loses its interaction with the binding site residues (Table 1). A total of 11 hydrogen bonds (nine direct and two water-mediated) are involved in the structural stabilization of the complex H5-N23G in BM2 (Fig. 6B). The interaction energy between the receptor and the interacting residues Tyr-91, Val-131, Ser-132, Ser-133, Ser-141, His-179, Glu-186, Asn-182, Lys-189, and Lys-219 has been computed and is shown in Fig. 4E. During the 10-ns simulation, the receptor loses its interactions with the binding site residues after 6 ns and is clearly seen in the interaction energy plot (Fig. 4E). An energy difference of 10 kcal/mol was noted between BM1 and BM2.

FIGURE 6.

FIGURE 6.

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Interactions between H5 and receptor H5-N23G at BM1 (A), H5-N23G at BM2 (B), and H5-N26G at BM1 (C).

H5 accommodates N26G in a single binding mode with the preferred glycosidic torsional angle of −70 and 50°, and the region is marked as AN23G-H5 (Fig. 2F). The preferred exocyclic torsional angle is 70°. A total of 11 hydrogen bonds (seven direct and four water-mediated) are involved in the structural stabilization of H5-N26G complex (Fig. 6C), and these interactions are given in Table 1. The interaction energy difference between H5-N23G and H5-N26G was 6.8 ± 0.11 kcal/mol, with H5-N23G having the minimum energy. The relative binding free energy between the complexes was calculated to be 2.4 ± 6.1 kcal/mol using MM-PBSA, where H5-N23G is the minimum when compared with H5-N26G. Based on atomistic level interactions, interaction energy analysis, and binding free energy of the complexes, it is concluded that, in the receptor activity of sialyldisaccharides, “N23G” is a better receptor for H5 when compared with N26G.

N23G and N26G at the Binding Pocket of H9, Conformation and Interaction Energy

The analysis on the MD simulation trajectories of H9-N23G complex clearly shows that a single binding mode (BM1) is plausible for N23G and the preferred glycosidic linkage torsional angle is ΦN23G-H9 and ΨN23G-H9 at −70 and 0° (Fig. 2G). The interactions between the binding site residues and the receptor are listed in Table 1. In this binding mode, most of the functional groups of sialic acid form hydrogen bonds with the binding site residues of H9. Galactose residue makes a single hydrogen bond with the binding site residue Gly-215. A total of nine hydrogen bonds (eight direct and one water mediated) stabilize the H9-N23G complex (Fig. 7A). The interaction energy plot between N23G and the interacting binding site residues Thr-129, Ser-130, Ser-131, Tyr-91, Asn-173, and Gly-215 of H9 is shown in Fig. 4G, and it reflects a single binding mode.

FIGURE 7.

FIGURE 7.

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Interactions between H9 and receptor H9-N23G (BM1) (A) and H9-N26G (BM1) (B).

The glycosidic torsional distribution of N26G in the H9-N26G complex indicates a flexible binding mode for N26G at the binding site of H9 (Fig. 2H) and is reflected in the interaction energy plot (Fig. 4H). A maximum of seven hydrogen bonds stabilize the structure of this complex (Fig. 7B). The interaction energy difference between H9-N23G complex and H9-N26G complex was about 2.3 ± 0.1 kcal/mol, with H9-N23G being the minimum energy. The relative binding free energy difference of 0.1 ± 4.6 kcal/mol has been observed through MM-PBSA calculations where H9-N26G was marginally minimum over H9-N23G. The above results indicate that H9 can bind to N23G and N26G with approximately equal potential.

DISCUSSION

The binding modes of the glycans, especially the sialyldisaccharides, greatly influence the selectivity of the hemagglutinin recognition and specificity toward glycan receptors. 10-ns MD simulations carried out on H1, H3, H5, and H9 complexed with N23G and N26G revealed the different binding modes for receptors in the viral binding pocket. Careful analysis of the sialyldisaccharide topology in the binding site of HAs and the interaction energy between the sialyldisaccharides and the binding site residues of HA are carried out. Also, relative binding free energies of the complexes are found using MM-PBSA calculations.

To validate the MD simulation results, the crystallographic structures of the HAs (Protein Data Bank codes 1RV0, 1MQM, 1JSN, and 1JSH) are analyzed for hydrogen bonding interactions between receptor and the proteins. The analysis reveals that at the binding pocket of H1, H3, H5, and H9, the bound sialyldisaccharide has 6, 8, 11, and 4 direct hydrogen bonds, respectively. However, MD simulation on the H1-N23G complex reveals two plausible binding modes with 10 hydrogen bonds in BM1 and 9 hydrogen bonds in BM2. A strong hydrogen bonding network is formed between disaccharides and the binding site residues Tyr, Thr, Ala, and Gln, and these hydrogen bonds stabilize the complex structure. Similar hydrogen bonding network has been observed earlier by Nunthaboot et al. (57) using a 6.5-ns MD on H1 of 1918, 1930, 2005, and 2009 epidemics. A 4-ns MD simulation by Nadtanet et al. (58) on the complex of sialopentasaccharide with hemagglutinin H1 (Protein Data Bank code 1RVT) found a single rigid conformation for N26G. The reported glycosidic torsional angle for Φ is −68°. In our simulations, in the strong binding mode BM1, the value is Φ −70° and it closely matches with the earlier reported result. In addition to BM1, another binding mode is also predicted by our 10-ns MD simulations with the glycosidic torsional angle of −150 and 50°. The glycosidic torsional angle for N23G in the crystal structure of H3-N23G complex is −174 and −2°, with eight hydrogen bonds, and the preferred glycosidic torsion found for N23G from 10-ns MD simulation is −90 and −50°, with 12 hydrogen bonds. This indicates that the solution state binding mode of sialyldisaccharides differs from the crystal structure binding mode. For the H3-N26G complex, we propose two binding modes with 10 direct hydrogen bonding interactions in each mode. In the crystal structure of the H5-N23G complex, the glycosidic torsional angle is −170 and −24°. Our simulation of a 10-ns duration predicted two possible binding modes with the glycosidic torsional angles of −120 and −50° and −70 and 0°. The best binding mode (−120 and −50°) falls in the same region as that of crystal structure. For H5-N26G, the MD simulation predicted a single binding mode with 11 hydrogen bonds. In the crystal structure, H9-N23G has the glycosidic torsional angles of −70 and −22°. In our MD simulation, the preferred glycosidic torsional angle is −70 and 0°. This indicates that the solution state conformation closely matches the crystal structure, and an increase in the number of hydrogen bonding interactions between the sugar and the binding site residues is observed in our simulation model. When H9 accommodates N26G, the conformation around the glycosidic torsional angle is widely distributed (Fig. 2H). The interaction energy is marginally higher (2.3 ± 0.08 kcal/mol) when compared with the H3-N23G complex. This indicates that H9 can bind with N23G and N26G in almost equal probability. The consolidated results of 10-ns MD simulations on the complexes of sialyldisaccharides with H1, H3, H5, and H9 are given in Table 2. This table details the different binding modes, the number of hydrogen bonds, and the relative interaction energy values for HA-sialyldisaccharide complexes. The binding free energy of the water molecule involved in water-mediated hydrogen bonding interactions has been calculated, and it varies from −3.1 to −4.1 kcal/mol (Table 1). The full structures of equilibrated complexes in the best binding modes are given in supplemental Fig. S2.

TABLE 2.

Possible binding modes, interaction energy, binding free energy, and the receptor specificity of the complexes

D indicates direct hydrogen bond, and W indicates water-mediated hydrogen bond.

S. no. Complex Possible binding modes No. of hydrogen bonds
 Relative interaction energy between the complexes in best binding mode Binding free energy of the complexes using MM-PBSA Relative binding free energy of the complexes using MM-PBSA Receptor specificity
BM1 BM2
kcal/mol kcal/mol kcal/mol
1. H1-N23G BM1, BM2 10 (6D, 4W) 9 (5D, 4W) 19.0 ± 0.28 −8.9 ± 5.5 5.6 ± 5.5 N26G > N23G
H1-N26G BM1, BM2 14 (8D, 6W) 11 (6D, 5W) 0.0 ± 0.51 −14.5 ± 3.8 0.0 ± 3.8
2. H3-N23G BM1 12 (12D) − 0.0 ± 0.08 −30.3 ± 5.4 0.0 ± 5.4 N23G > N26G
H3-N26G BM1, BM2 10 (10D) 10 (10D) 2.1 ± 0.32 −27.3 ± 4.7 3.0 ± 4.7
3. H5-N23G BM1, BM2 14 (9D, 5W) 11 (9D, 2W) 0.0 ± 0.47 −31.6 ± 5.5 0.0 ± 5.5 N23G > N26G
H5-N26G BM1 11 (7D, 4W) − 6.8 ± 0.11 −29.2 ± 6.1 2.4 ± 6.1
4. H9-N23G BM1 9 (8D, 1W) − 0.0 ± 0.05 −19.2 ± 4.6 0.1 ± 4.6 N23G > N26G
H9-N26G BM1 7 (7D) − 2.3 ± 0.08 −19.3 ± 3.6 0.0 ± 3.6
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Based on relative interaction energy between the complexes, it is concluded that, except for H1, N23G is a better receptor for H3, H5, and H9 when compared with N26G. It is also concluded that based on direct and water-mediated hydrogen bonding interactions between the binding site residues and sialyldisaccharides, the order of specificity for N23G is H3 > H5 > H9 > H1 and for N26G is H1 > H3 > H5 > H9. The overall trends observed in energy calculation by NAMD are also reflected in the MM-PBSA calculations. In this study, we provide the atomistic level interactions between HA and sialyldisaccharides, N23G and N26G. Understanding the recognition specificities based on atomistic level interactions will help in developing more efficient antiviral drugs and designing carbohydrate-based ligands to inhibit the pathogenic processes.

Acknowledgment

We acknowledge the use of the Bioinformatics Center at the Department of Physics, Manonmaniam Sundaranar University, which is funded by Department of Biotechnology Grant BT/BI/25/001/2006.

Inline graphic

This article contains supplemental Figs. S1 and S2.

5
The abbreviations used are:
NA
neuraminidase
MD
molecular dynamics
N23G
Neu5Acα(2–3)Gal
N26G
Neu5Acα(2–6)Gal
NAMD
Nanoscale Molecular Dynamics.

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