theoretical investigation on the binding specificity of

Theoretical Investigation on the Binding Specificity ofSialyldisaccharides with Hemagglutinins of Influenza A Virusby Molecular Dynamics Simulations□S

Received for publication, March 1, 2012, and in revised form, July 11, 2012 Published, JBC Papers in Press, July 30, 2012, DOI 10.1074/jbc.M112.357061

Thanu R. K. Priyadarzini‡1, Jeyasigamani F. A. Selvin‡2, M. Michael Gromiha§, Kazuhiko Fukui¶3,and Kasinadar Veluraja‡3,4

From the ‡Department of Physics, Manonmaniam Sundaranar University, Tirunelveli, Tamilnadu 627 012, India, the §Departmentof Biotechnology, Indian Institute of Technology Madras, Tamilnadu 600 036, India, and the ¶Computational Biology ResearchCenter, National Institute of Advanced Industrial Science and Technology, Tokyo 135-0064, Japan

Background: Recognition of terminal sialyldisaccharides by influenza A hemagglutinin initiates the infection process ofinfluenza.Results: MD simulations on sialyldisaccharide-hemagglutinin (H1, H3, H5, and H9) complexes reveal the molecular basis ofspecific recognition.Conclusion:The order of the binding specificity ofNeu5Ac�(2–3)Gal andNeu5Ac�(2–6)Gal isH3�H5�H9�H1andH1�H3 � H5 � H9, respectively.Significance: The insights from this study will help in designing carbohydrate-based therapeutics against influenza viralinfections.

Recognition of cell-surface sialyldisaccharides by influenza Ahemagglutinin (HA) triggers the infection process of influenza.The changes in glycosidic torsional linkage and the receptorconformations may alter the binding specificity of HAs to thesialylglycans. In this study, 10-ns molecular dynamics simula-tions were carried out to examine the structural and dynamicbehavior of the HAs bound with sialyldisaccharides Neu5Ac�(2–3)Gal (N23G) and Neu5Ac�(2–6)Gal (N26G). The analysis of theglycosidic torsional angles and the pair interaction energybetween the receptor and the interacting residues of the bindingsite reveal that N23G has two bindingmodes for H1 andH5 anda single bindingmode for H3 andH9. For N26G, H1 andH3 hastwo binding modes, and H5 and H9 has a single binding mode.The direct and water-mediated hydrogen bonding interactionsbetween the receptors andHAsplay dominant roles in the struc-tural stabilization of the complexes. It is concluded from pairinteraction energy and Molecular Mechanic-Poisson-Boltz-mann Surface Area calculations that N26G is a better receptorforH1when comparedwithN23G.N23G is a better receptor forH5when comparedwithN26G.However, H3 andH9 can recog-nize N23G and N26G in equal binding specificity due to themarginal energy difference (≈2.5 kcal/mol). The order of bind-ing specificity ofN23G isH3>H5>H9>H1andN26G isH1>H3 > H5 > H9, respectively. The proposed conformationalmodelswill behelpful indesigning inhibitors for influenza virus.

Influenza is a zoonotic disease caused by RNA viruses of theOrthomyxoviridae family. Influenza viruses are roughly spher-ical in shape with glycoprotein spikes on their cell surface, andtheir genome consists of eight RNA fragments that encode 10proteins. Among the surface glycoproteins, hemagglutinin(HA) and neuraminidase (NA)5 are critical for the biology ofinfluenza viruses. Based on the serological differences, influ-enza viruses are divided into three types: A, B, and C (1–7). Ofthe three types, influenza A viruses can infect a variety of ani-mals, including poultry, human, pigs, horse, murine mammals,and carnivore animals (8–10). The spikes of HA bind to sialicacids that are found on the surface of the host cell membraneand mediate virus attachment to target cells (11–15). NAcleaves the�-glycosidic linkage between the terminal sialic acidand the penultimate sugar residue, facilitating elution of virusprogeny from infected cells and preventing self-aggregation ofthe virus. Depending on the type of glycoproteins, HA and NA,influenzaA viruses are further classified into different subtypes.So far, 16 different subtypes of HA and 9 subtypes of NA havebeen found. Of the 16 subtypes of HA, most are isolated fromavian species and a few from equine (H3 and H7), seals (H3, H4and H7), whales (H1 and H13), and swine (H1, H3 and H9)(16–22). Influenza A subtypes H1, H2, and H3 had caused fivepandemics in last century, viz. Spanish 1918 (H1), Asian 1957(H2), Hong Kong 1968 (H3), Russian 1971 (H1), and Mexicanflu 2009 (H1) (23–26). The receptor binding specificity of influ-enza viruses is related to the recognition of cellular sialic acidsby the viral hemagglutinin (27–30).Sialic acids are significant sugar molecules typically found at

the outermost ends of N-glycans, O-glycans, or glycosphingo-lipids. They exist in diversified forms that arise from the mod-

□S This article contains supplemental Figs. S1 and S2.1 Recipient of Department of Biotechnology Grant BT/PR4251/BID/07/2003.2 Recipient of Department of Science and Technology Grant SR/S0/BB-53/

2003 and a grant from Council of Scientific and Industrial Research.3 Supported by the Department of Biotechnology, India, and National Insti-

tute of Advanced Industrial Science and Technology, Japan CollaborativeProject BT/BI/14/028/2007.

4 To whom correspondence should be addressed. Tel.: 91-462-2336768; Fax:91-462-2334363; E-mail: [email protected].

5 The abbreviations used are: NA, neuraminidase; MD, molecular dynamics;N23G, Neu5Ac�(2–3)Gal; N26G, Neu5Ac�(2– 6)Gal; NAMD, NanoscaleMolecular Dynamics.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 41, pp. 34547–34557, October 5, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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ifications of 5th position in neuraminic acid and additional sub-stitutions at the hydroxyl group on the 4th, 7th, 8th, and 9thcarbon, and the other diversified forms are generated by thelinkages from the 2nd carbon to the penultimate sugar. Themost common linkages are Neu5Ac�(2–3)Gal (N23G) andNeu5Ac�(2–6)Gal (N26G). These structural diversities ofsialic acid can govern the recognition by a variety of sialic acid-binding lectins such as influenza virus hemagglutinin. Thereceptor specificity of influenza viruses is defined in terms oftheir recognition of sialic acid species (Neu5Ac, Neu5Gc,Neu4,5Ac2, etc.), and the type of glycosidic linkage betweensialic 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 sialicacid (34–37). Influenza A viruses isolated from human andavian recognize N26G andN23G, respectively. Swine influenzavirus can bind with both N23G and N26G.A single amino acid change in the receptor-binding site of

hemagglutinin of influenzaA virus alters the binding specificityfrom N23G to N26G (38, 39). It has been reported by Tauben-berger and co-workers (40) that mutation at positions 190 and225 (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 bindingwith proteins (41–45). In this study, 10-ns MD simulations arecarried out for the complexes of H1-N23G, H1-N26G,H3-N23G, H3-N26G, H5-N23G, H5-N26G, H9-N23G, andH9-N26G to understand the binding specificity of sialyldisac-charides with hemagglutininH1, H3, H5, andH9 of influenzaAvirus, and the probable binding models are proposed for theabove complexes. The pair interaction energies of the com-plexes are calculated using Nanoscale Molecular Dynamics(NAMD).Hydrogenbonding patterns,which stabilize the com-plexes, are found, and the binding free energy due to the watermolecule mediating the interaction is also calculated usingNAMD for each water-mediated hydrogen-bonding interac-tion. The binding free energy of the complexes is calculatedusing MM-PBSA calculation.

COMPUTATIONAL DETAILS

The molecular structures of N23G and N26G along with theglycosidic torsional angles� and� are shown in Fig. 1,A andB.The geometry of N23G and N26G is generated using the stan-dard bond length, bond angles, and torsional angles (46, 47).The three-dimensional coordinates of influenza A hemag-glutinins were taken from the Protein Data Bank of ResearchCollaboratory for Structural Bioinformatics. In this study,we have used the HAs of influenza A strains H1, H3, H5, andH9 and the respective Protein Data Bank codes are 1RV0,1MQM, 1JSN, and 1JSH (48–50). The number of aminoacids in H1, H3, H5, and H9 are 324, 318, 321, and 317respectively. N23G and N26G are modeled into the bindingsite of H1, H3, H5, and H9. The force field parameters gaff(51) and ff99 (52) are used to prepare the input parametersfor sugar and protein, respectively. Care is taken to neutral-ize the entire protein-sugar complex. The total system issolvated with TIP3P water molecules using solvents library

of AMBER9. The temperature of the ensemble is kept at 300K, and constant pressure is maintained. The cutoff for thenonbonding interactions was set to 10 Å. We have usedsander of AMBER9 (53) to perform the MD simulation. MDsimulations of 10-ns duration was carried out for thecomplexes H1-N23G, H1-N26G, H3-N23G, H3-N26G, H5-N23G, H5-N26G, H9-N23G, and H9-N26G. The trajectoriesare collected over every picosecond, and a total of 10,000 struc-tures are collected for each simulation. To get insights on theatomistic level interactions between the receptor sugar residueand HA protein, graphical software VMD (54) is used alongwith the FORTRAN programs developed in-house. The inter-action energy between the interacting amino acid residues andthe receptor sugar residue is computed by NAMD (55). Thebinding free energy between the protein and the receptoris calculated using MM-PBSA module of AmberTools.MOLSCRIPT (56) is used to render the molecular interactions.

RESULTS

The results of 10-ns MD simulations carried out for eightprotein-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 devel-oped FORTRAN programs. Information about conformationalflexibility of the receptors (N23G and N26G), atomistic levelinteraction between functional groups of receptor (carboxylate,acetyl, glycerol side chain, hydroxyl, and hydroxymethyl), andthe binding site residues were obtained to gain insights on thebinding affinity of the receptors with different hemagglutininsof influenza A virus. The possible conformations of sialyldisac-charides were deduced from the glycosidic torsional map, andthe associated interaction energies of theHA-sialyldisaccharidecomplexes were calculated. The interaction patterns wereproposed based on rigorous analysis of the MD simulationtrajectories and superimposed trajectories of various frames

FIGURE 1. Schematic representation. A, Neu5Ac�(2–3)Gal (�N23G � C1-C2-O2-C3; �N23G � C2-O2-C3-H3) (N23G). B, Neu5Ac�(2– 6)Gal (N26G) (�N26G �C1-C2-O2-C6; �N26G � C2-O2-C6-H61; �N26G � O2-C6-C5-O5 (H61 is thehydrogen that makes an angle of H61-C6-C5-O5 � 120° when �N26G � 0°);�n � C5-N5-C10-C11; �1 � C5-C6-C7-C8; �2 � C6-C7-C8-C9; �3 � C7-C8-C9-O9; �4 � C1-O5-C5-C6).

Binding Specificity of Influenza toward Sialyldisaccharides

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having similar conformation for sialyldisaccharides. The rel-ative binding free energies of the sialyldisaccharides-proteincomplexes were calculated using MM-PBSA module ofAmberTools. The binding free energy of the water molecule,which is involved in water mediation, was calculated usingNAMD. The conformations of the receptors N23G andN26G at the binding site of hemagglutinin were dictated byglycosidic conformational torsional angles denoted as � and�. In addition to the glycosidic torsional angles, N26G pos-sesses an exocyclic torsional angle (�). The energy of thecomplexes of each hemagglutinin with N23G and N26G canbe compared with each other because the number of atomsin each simulation of similar hemagglutinin was the same.Conformation of Sialyldisaccharides N23G and N26G at the

Binding Pocket of HAs—The conformational flexibilities of thereceptors N23G and N26G were examined in terms of acet-amido group conformation (�n � C5-N5-C10-C11), glycerolside chain conformation (�1 � C5-C6-C7-C8; �2 � C6-C7-C8-C9; and �3 � C7-C8-C9-O9) of sialic acid, and the hydroxy-methyl group conformation (�4 � C4-C5-C6-O6) of galactose.The 10-ns MD simulations on the complexes indicated that inall the HA-N23G complexes �n preferred the trans conforma-tion;�1 preferred trans for the complexesH1-N23G,H5-N23G,and H9-N23G. For H3-N23G complex, �1 preferred trans andg�. �2 preferred g� for H1-N23G, H5-N23G, and H9-N23Gcomplexes and trans for H3-N23G complex. The terminalhydroxyl group conformation �3 preferred trans conformationfor H1-N23G and H3-N23G complexes, whereas both transand g� conformations are preferred for H5-N23G andH9-N23G complexes. �4 of galactose preferred g� in all thecomplexes.In HA-N26G complexes, �n and �1 preferred trans confor-

mation in all the complexes. �2 preferred trans conformationfor H1-N26G and H3-N26G and both trans and g� conforma-tions for H5-N26G and H9-N26G complexes. In all the com-plexes �3 preferred trans and g� conformations.The overall structural flexibility of the HA-sialyldisaccharide

complexes was measured in terms of root mean square devia-tion of backbone, and the root mean square deviation trajecto-ries are given as supplemental Fig. S1.N23G and N26G at the Binding Pocket of H1, Conformation,

and Interaction Energy—The conformational features of thereceptor N23G and N26G in the binding site of H1 hemagglu-tininwere computed, and the conformer distributions based onglycosidic torsions are given in Fig. 2,A and B, respectively. It isevident from Fig. 2A that N23G exists in three different confor-mational regions inside the binding pocket of H1, and the gly-cosidic torsional values (�N23G-H1 and �N23G-H1) of theseregions are AN23G-H1 (�150 and �30°), BN23G-H1 (�100 and�50°), and CN23G-H1 (�70 and 0°) with respective populationpropensities of 37, 35, and 16%. The three regions are denotedas BM1, BM2, and BM3, respectively. When N23G exists inbindingmode 1 (BM1), the carboxylate group formedhydrogenbonds with the hydroxyl of Thr-133, backbone amide group ofAla-134, and side chain nitrogen of Gln-223. Also, the carbox-ylate group formed a water-mediated hydrogen bond with sidechain hydroxyl of Ser-142. O4 hydroxyl of sialic acid interactedwith 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 groupof Tyr-91. The glycerol side chain hydroxyl oxygens O8 andO9formed either a direct or water-mediated hydrogen bond withthe side chain oxygen of Asp-187. The hydroxymethyl group ofgalactose residue made a hydrogen bond with the backboneamide group of Ala-224. In BM1, a total of 10 hydrogen bonds(six direct and four water-mediated) are involved in the struc-tural stabilization of the receptor at the binding site of H1 (Fig.3A), and the interactions are given in Table 1. In binding mode2 (BM2), the following interactions were observed between thereceptor and protein. The interactions between the binding siteresidues and the carboxylate group, O8 and O9 hydroxyl oxy-gens of glycerol side chain, were similar to those of BM1. How-ever, the hydroxymethyl group of galactose made a hydrogenbond 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 ofH1-N23G in BM2 (Fig. 3B). At the CN23G-H1 region (BM3), theinteraction between binding site residues and sialic acid wassimilar as was that of BM2. However, the second residue galac-

FIGURE 2. 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), andH9-N26G (H).




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tose did not make any hydrogen bonds with the binding siteresidues. Interaction energy between the receptor and the bind-ing site residues ofH1 (Tyr-91, Arg-130, Val-132, Thr-133, Ala-134, His-180, Asp-187, Gln-223, Ala-224, andGly-225) was cal-culated usingNAMDand plottedwith respect to time as shownin Fig. 4A. It is evident from Fig. 4A that interaction energy fallsinto three distinct regions, and this substantiates the abovebinding modes. In the interaction energy plot, the energy wasminimum for 0–3500 ps and corresponded to BM1. The sec-ond dominant region was mainly observed between 5000 and7500 ps, and it corresponded to BM2. The interaction energygraph clearly confirmed that although H1 can accommodateN23G in three distinct modes, BM1 was the best binding modefor H1.In the MD trajectories of the H1-N26G complex, the disac-

charide N26G preferred two distinct conformations AN26G-H1and BN26G-H1 with the glycosidic torsional angle (�N26G-H1 and�N26G-H1) values of �150 and 70° and �70 and 70° with thepopulation propensities of 35 and 60%, respectively, andthe exocyclic torsional angle predominantly preferred 70°. Inthe BN26G-H1 (BM1) region, N26G was buried well within thebinding site and made a cluster of direct and water-mediatedhydrogenbondswith the binding site residues. The interactionsof sialic acid with the binding site were as follows: carboxylate

groupmade 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; O4hydroxyl of sialic acidwith a carbonyl of Arg-130 throughwatermediation; acetamido group forms hydrogen bond with car-bonyl of Val-132; O8 hydroxyl with polar side chain of Tyr-91;O9 hydroxyl with side chain nitrogen of His-180; O9 throughwater mediation with a side chain hydroxyl of Gln-223, amidenitrogen of Ala-224 and carbonyl of Gly-225. Interactions ofgalactose with the binding site residues are given below:O3 andO4 hydroxyl with carbonyl of Gly-222; O3/O2 hydroxyl withLys-219 through water mediation. These interactions are givenin Table 1 (Fig. 3C). A total of 14 hydrogen bonds (eight directand six water-mediated) stabilize the complex in this bindingmode (BM1). When the N26G prefers AN26G-H1 (BM2), theinteraction between sialic acid and the binding site residues aresimilar to BM1. However, the galactose residue does not inter-act with the binding site residues. A total of 11 hydrogen bonds(six direct and five water-mediated) are involved in the struc-tural stabilization of this complex in BM2 (Fig. 3D). These bind-ing modes are reflected in interaction energy plot of H1-N26G(Fig. 4B), which exhibits two distinct modes. The NAMD-cal-culated interaction energy difference between H1-N23G andH1-N26G was 19.0 � 0.28 kcal/mol, with H1-N26G being the

FIGURE 3. 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).




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minimum. The binding free energy of the complex H1-N23Gand H1-N26G was calculated by using MM-PBSA calculationand the results were that the relative binding free energy differ-ence between the two complexes was 6.4 � 5.6 kcal/mol, withH1-N26G is beingminimum. The atomistic level of interactionbetween the binding site residues and the receptor, the interac-tion energy analysis of the complexes, and the binding energybetween the complexes show that N26G is the better receptorfor H1 when compared with N23G.N23G and N26G at the Binding Pocket of H3, Conformation

and Interaction Energy—The conformational flexibility of thereceptor N23G in the complex H3 is shown in Fig. 2C, and itshows that a single glycosidic torsional region (�N23-H3 and

�N23-H3) is preferred and is denoted as AN23G-H3. The pre-ferred glycosidic torsional angle was �90 and �50°. Theatomistic level interactions between the receptor and thebinding site residues were analyzed and are given in Table 1.A cluster of 12 direct hydrogen bonds were involved in sta-bilizing the H3-N23G complex. The flexibility of the recep-tor N23G was limited to a single rigid conformation, and thiswas due to the interactions of both the residues of N23GwithH3 (Fig. 5A). The interaction energy between interactingamino acid residues (Tyr-90, Gly-127, Ser-128, Ser-129,Asn-137, His-175, Glu-182, Gly-217, and Gln-218) of H3 andN23G has been computed, and a single binding mode isreflected (Fig. 4C).

TABLE 1Interactions between receptors (N23G and N26G) and the active site residues of H1, H3, H5, and H9 hemagglutinins

Receptor Binding modesReceptoratom

Protein atomsin H1

Binding freeenergy due to

water mediationProtein atoms

in H3

Binding freeenergy of due towater mediation

Protein atomsin H5


water mediationProtein atoms

in H9


water mediation

kcal/mol kcal/mol kcal/mol kcal/molN23G 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 NGln-223 NE2 � Ser-129 OG � Ser-133 OG � Ser-131 OGWAT-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 � 0.5N5 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 ND2His-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 � 0.2Binding 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 OGWAT-Ser-142 OG �3.3 � 0.5 Ser-129 OG � Ser-133 OG � Ser-131 OGGln-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 OE2WAT-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 � 0.8WAT-Gly-225 N �3.6 � 0.8

O2B � � Gly-217 O � � � �O3B � � Gly-217 O � � � �O4B � � � � � � �O6B � � � � � � �




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In the H3-N26G complex, the receptor has two modes ofbinding via BM1 (AN26G-H3) with the glycosidic torsion of �70and 50° and BM2 (BN26G-H3) with�90 and�50° as given in Fig.2D. The respective population propensities for the two bindingmodes are 42 and 31%. The exocyclic torsional value is 70°. Theatomistic level interaction between the receptor N26G and thebinding site residues in BM1 are given in Table 1. A total of 10hydrogen bonds are involved in the structural stabilization ofthe complex H3-N26G at BM1 as shown in Fig. 5B. In BM2,both the residues were interacting with the binding site resi-dues of H3. In BM1, the O2 andO3 hydroxyl of galactose inter-

acts with H3 and in BM2, and the O3 andO4 hydroxyl of galac-tose interacts with H3 (Table 1), and this is reflected in theinteraction energy plot (Fig. 4D). The atomistic level interac-tion in BM2 is shown in Fig. 5C. The difference in interactionenergy between H3-N23G and H3-N26Gwas calculated, and itsuggested that H3-N26G has 2.1� 0.32 kcal/mol higher energythan H3-N23G. This energy difference can be attributed to thedifferences in the number of hydrogen bonds formed betweenthe receptor and protein complex. The relative binding freeenergy of the complexes was calculated using the MM-PBSAmodule and was 3.0 � 4.7 kcal/mol with H1-N23G being the

FIGURE 4. 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), andH9-N26G (H).




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minimum. Based on the atomistic level interactions betweenthe receptor and the binding site residues of H3, interactionenergy analysis, and the binding free energy of the complexes, itwas concluded that N23G is the better receptor for H3 whencompared with N26G.N23G and N26G at the Binding Pocket of H5, Conformation

and InteractionEnergy—The analysis on the complex dynamicsof H5-N23G indicates that H5 can accommodate N23G in twodifferent modes denoted as regions AN23G-H5 (BM1) andBN23G-H5 (BM2), and the respective preferred glycosidic tor-sional angles for �N23G-H5 and �N23G-H5 are �120 and �50°and �70 and 0° (Fig. 2E). In BM1, sialic acid makes eight directhydrogen bonds and three water-mediated hydrogen bondsand galactose makes one direct hydrogen bond and two water-mediated hydrogen bonds with the binding site residues (Table1). 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 thehydroxymethyl group of galactose loses its interaction with thebinding site residues (Table 1). A total of 11 hydrogen bonds(nine direct and two water-mediated) are involved in the struc-tural stabilization of the complex H5-N23G in BM2 (Fig. 6B).The interaction energy between the receptor and the interact-ing residues Tyr-91, Val-131, Ser-132, Ser-133, Ser-141, His-179, Glu-186, Asn-182, Lys-189, and Lys-219 has been com-puted and is shown in Fig. 4E. During the 10-ns simulation, thereceptor loses its interactions with the binding site residuesafter 6 ns and is clearly seen in the interaction energy plot (Fig.4E). An energy difference of 10 kcal/mol was noted betweenBM1 and BM2.H5 accommodates N26G in a single binding mode with the

preferred glycosidic torsional angle of �70 and 50°, and theregion is marked as AN23G-H5 (Fig. 2F). The preferred exocyclictorsional angle is 70°. A total of 11 hydrogen bonds (seven directand four water-mediated) are involved in the structural stabili-zation of H5-N26G complex (Fig. 6C), and these interactionsare given in Table 1. The interaction energy difference betweenH5-N23G and H5-N26G was 6.8 � 0.11 kcal/mol, withH5-N23G having the minimum energy. The relative bindingfree energy between the complexes was calculated to be 2.4 �6.1 kcal/mol using MM-PBSA, where H5-N23G is the mini-mumwhen compared with H5-N26G. Based on atomistic level

interactions, interaction energy analysis, and binding freeenergy of the complexes, it is concluded that, in the receptoractivity of sialyldisaccharides, “N23G” is a better receptor forH5 when compared with N26G.N23G and N26G at the Binding Pocket of H9, Conformation

and Interaction Energy—The analysis on the MD simulationtrajectories of H9-N23G complex clearly shows that a singlebinding mode (BM1) is plausible for N23G and the preferredglycosidic linkage torsional angle is �N23G-H9 and �N23G-H9 at�70 and 0° (Fig. 2G). The interactions between the binding siteresidues and the receptor are listed in Table 1. In this bindingmode, most of the functional groups of sialic acid form hydro-gen bonds with the binding site residues of H9. Galactose resi-duemakes a single hydrogen bondwith the binding site residueGly-215. A total of nine hydrogen bonds (eight direct and onewater mediated) stabilize the H9-N23G complex (Fig. 7A). Theinteraction energy plot between N23G and the interactingbinding 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 asingle binding mode.The glycosidic torsional distribution of N26G in the

H9-N26G complex indicates a flexible binding mode for N26Gat the binding site of H9 (Fig. 2H) and is reflected in the inter-action energy plot (Fig. 4H). A maximum of seven hydrogenbonds stabilize the structure of this complex (Fig. 7B). Theinteraction energy difference between H9-N23G complex andH9-N26G complex was about 2.3 � 0.1 kcal/mol, withH9-N23Gbeing theminimumenergy. The relative binding freeenergy difference of 0.1 � 4.6 kcal/mol has been observedthroughMM-PBSA calculations where H9-N26G was margin-ally minimum over H9-N23G. The above results indicate thatH9 can bind to N23G and N26G with approximately equalpotential.

DISCUSSION

The binding modes of the glycans, especially the sialyldisac-charides, greatly influence the selectivity of the hemagglutininrecognition and specificity toward glycan receptors. 10-ns MDsimulations carried out onH1, H3, H5, andH9 complexed withN23G and N26G revealed the different binding modes forreceptors in the viral binding pocket. Careful analysis of thesialyldisaccharide topology in the binding site of HAs and

FIGURE 5. Interactions between H3 and receptor H3-N23G at BM1 (A), H3-N26G at BM1 (B), and H3-N26G at BM2 (C).




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the interaction energy between the sialyldisaccharides and thebinding site residues of HA are carried out. Also, relative bind-ing free energies of the complexes are found using MM-PBSAcalculations.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 interac-tions between receptor and the proteins. The analysis revealsthat at the binding pocket of H1, H3, H5, and H9, the boundsialyldisaccharide has 6, 8, 11, and 4 direct hydrogen bonds,respectively. However, MD simulation on the H1-N23G com-plex reveals two plausible binding modes with 10 hydrogenbonds in BM1 and 9 hydrogen bonds in BM2. A strong hydro-gen bonding network is formed between disaccharides and the

binding site residuesTyr, Thr,Ala, andGln, and these hydrogenbonds stabilize the complex structure. Similar hydrogen bond-ing network has been observed earlier byNunthaboot et al. (57)using a 6.5-nsMDonH1 of 1918, 1930, 2005, and 2009 epidem-ics. A 4-ns MD simulation by Nadtanet et al. (58) on the com-plex of sialopentasaccharide with hemagglutinin H1 (ProteinData Bank code 1RVT) found a single rigid conformation forN26G. The reported glycosidic torsional angle for � is �68°. Inour simulations, in the strong bindingmodeBM1, the value is��70° and it closely matches with the earlier reported result. Inaddition to BM1, another bindingmode is also predicted by our10-ns MD simulations with the glycosidic torsional angle of�150 and 50°. The glycosidic torsional angle for N23G in thecrystal structure of H3-N23G complex is �174 and �2°, with

FIGURE 6. Interactions between H5 and receptor H5-N23G at BM1 (A), H5-N23G at BM2 (B), and H5-N26G at BM1 (C).

FIGURE 7. Interactions between H9 and receptor H9-N23G (BM1) (A) and H9-N26G (BM1) (B).




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eight hydrogen bonds, and the preferred glycosidic torsionfound for N23G from 10-ns MD simulation is �90 and �50°,with 12 hydrogen bonds. This indicates that the solution statebinding mode of sialyldisaccharides differs from the crystalstructure binding mode. For the H3-N26G complex, we pro-pose two binding modes with 10 direct hydrogen bondinginteractions in each mode. In the crystal structure of theH5-N23G complex, the glycosidic torsional angle is �170 and�24°. Our simulation of a 10-ns duration predicted two possi-ble binding modes with the glycosidic torsional angles of �120and �50° and �70 and 0°. The best binding mode (�120 and�50°) falls in the same region as that of crystal structure. ForH5-N26G, theMD simulation predicted a single binding modewith 11 hydrogen bonds. In the crystal structure, H9-N23G hasthe glycosidic torsional angles of �70 and �22°. In our MDsimulation, the preferred glycosidic torsional angle is �70 and0°. This indicates that the solution state conformation closelymatches the crystal structure, and an increase in the number ofhydrogen bonding interactions between the sugar and the bind-ing site residues is observed in our simulationmodel.WhenH9accommodates N26G, the conformation around the glycosidictorsional angle is widely distributed (Fig. 2H). The interactionenergy is marginally higher (2.3 � 0.08 kcal/mol) when com-pared with the H3-N23G complex. This indicates that H9 canbind with N23G and N26G in almost equal probability. Theconsolidated results of 10-nsMD simulations on the complexesof sialyldisaccharides with H1, H3, H5, and H9 are given inTable 2. This table details the different binding modes, thenumber of hydrogen bonds, and the relative interaction energyvalues for HA-sialyldisaccharide complexes. The binding freeenergy of the water molecule involved in water-mediatedhydrogen bonding interactions has been calculated, and it var-ies from �3.1 to �4.1 kcal/mol (Table 1). The full structures ofequilibrated complexes in the best binding modes are given insupplemental Fig. S2.Based on relative interaction energy between the complexes,

it is concluded that, except forH1, N23G is a better receptor forH3, H5, and H9 when compared with N26G. It is also con-cluded that based on direct and water-mediated hydrogenbonding interactions between the binding site residues and sia-lyldisaccharides, the order of specificity forN23G isH3�H5�H9 � H1 and for N26G is H1 � H3 � H5 � H9. The overalltrends observed in energy calculation by NAMD are alsoreflected in the MM-PBSA calculations. In this study, we pro-vide the atomistic level interactions between HA and sialyldi-saccharides, N23G and N26G. Understanding the recognition

specificities based on atomistic level interactions will help indeveloping more efficient antiviral drugs and designing carbo-hydrate-based ligands to inhibit the pathogenic processes.

Acknowledgment—We acknowledge the use of the BioinformaticsCenter at the Department of Physics, Manonmaniam SundaranarUniversity, which is funded by Department of Biotechnology GrantBT/BI/25/001/2006.

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TABLE 2Possible binding modes, interaction energy, binding free energy, and the receptor specificity of the complexesD indicates direct hydrogen bond, and W indicates water-mediated hydrogen bond.

S.no. Complex

Possible bindingmodes

No. of hydrogen bonds Relative interaction energybetween the complexes in

best binding mode

Binding free energyof the complexesusing MM-PBSA

Relative binding free energyof the complexesusing MM-PBSA

ReceptorspecificityBM1 BM2

kcal/mol kcal/mol kcal/mol1. 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 � 3.82. 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 � 4.73. 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 � 6.14. 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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Fukui and Kasinadar VelurajaThanu R. K. Priyadarzini, Jeyasigamani F. A. Selvin, M. Michael Gromiha, Kazuhiko

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

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