Binding of Influenza A Virus Hemagglutinin to the Sialoside Receptor

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J. Phys. Chem. B 2010, 114, 15700–15705

Binding of Influenza A Virus Hemagglutinin to the Sialoside Receptor Is Not Controlled by the Homotropic Allosteric Effect Toshihiko Sawada,*,†,‡ Dmitri G. Fedorov,† and Kazuo Kitaura†,§ Nanosystem Research Institute, National Institute of AdVanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan, Core Research for EVolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan, and Graduate School of Pharmaceutical Sciences, Kyoto UniVersity, Sakyo-ku, Kyoto 606-8501, Japan ReceiVed: July 23, 2010; ReVised Manuscript ReceiVed: October 12, 2010

Several sialoside receptors can bind to three active sites on influenza A viral hemagglutinin (HA), determining the mechanism of the virus and host cell binding. Optimization of complex structures at the molecular mechanics level shows an insignificant conformational change of HA between the isolated state and the complex with three sialosides. The energetic analysis of the HA (X-31, Aichi/2/1968)-sialoside complexes performed with quantum-mechanical calculations of the complex containing about 24 000 atoms at the FMO2-MP2/PCM/ 6-31G(d) level suggests that the binding of one, two, or three receptors has the same binding energy per sialoside, thus the trivalent HA-sialoside binding is not regulated by the sialoside homotropic allosteric effect. These results rationalize the experimentally reported simple binding mode for the trivalent HA-monovalent sialoside interaction in solution at equilibrium. Introduction

Computational Methods

Influenza A viral hemagglutinin (HA) has three active sites and can bind three R-sialoside receptors. HA is a spike glycoprotein expressed on the virion surface, consisting of the R-sialoside recognition domain HA1 and the membrane fusion domain HA2.1 The corresponding X-ray crystallographic structures have revealed that HA exists as a trimer of the HA1-HA2 unit possessing C3 symmetry and forming a cylindrical ectodomain of about 135 Å in the longest dimension. The shallow sialoside binding site is found on the top face of each HA1 domain surface, and the trimerization of HA1 allows the lipophilic HA2 trimer to achieve thermodynamic stability under mild pH conditions (Figure 1).2 The homotropic allosteric effect3,4 is an interesting phenomenon when the affinity for a ligand changes with the number of ligands (allosteric), and homotropic refers to the ligands being the same. This effect has wide implications on the binding process, and we investigate it for Neu5AcR2-6Galβ sialoside. Experimentally, the simple binding mode of soluble trivalent HA-monovalent sialoside association has been suggested by the nuclear magnetic resonance (NMR) titration study.5 However, its chemical origins and relation to multivalent HA-sialoside interaction are still unclear.

The model of the recombinant X-31 strain HA (Aichi/2/1968, human H3) in complex with human-type sialoside receptor Neu5AcR2-6Galβ was taken from an earlier work,6,7 where it was obtained starting from the corresponding X-ray crystallographic structure involving structural determined water.8-10 In this work, we also demonstrate the validity of our structures by performing classical molecular dynamics (MD) simulations. The modeling details are as follows. First, starting from the crystal structure of X-31 HA with three Neu5AcR2-3Galβ1-4Glc (PDB ID: 1HGG),9 we replaced these three sialosides by Neu5AcR2-6Galβ disaccharides, superposing the common Neu5AcR coordination. Next, we chose the initial conformation of the R2-6 glycoside bond so that the dihedral angles were: Neu C1-C2-Gal O6-C6 ) -57°, Neu C2-Gal O6-C6-C5 ) -154°, and Gal O6-C6-C5-O5 ) 62° (see Supporting Information (SI) Figure S1), using as a reference the X-31 HA-human R2-6 pentasialoside complex.10 Each Neu5AcR2-6Galβ has a negative charge from the carboxylate group at the C-1 position on the Neu5Ac residue (pKa ) 2.5-2.9),11 which is reasonable under the established mild HA-sialoside binding condition. The protonation of the polar amino acid side chains in the HA complex is as follows: all Arg form guanidinium, all Lys form ammonium, His75 in the HA1 domains forms imidazolium. His at positions 17 in HA1 and 142 in HA2 are neutral with the Nδ proton, and the other His are neutral with the Nε proton. All Asp and Glu form carboxylate. The prepared X-31 HA-(Neu5AcR2-6Galβ)3 complex involving crystallographically determined water was geometryoptimized (energy-minimized) by utilizing the class II force field12 implemented in the Discovery Studio program package.13 The minimization was performed using the adopted basis Newton-Raphson algorithm with the generalized Born implicit solvent (solvent dielectric constant ) 1, solvent generalized born dielectric constant ) 80) until the rms gradient fell below 0.0001

In the present study, we show that trivalent HA-monovalent sialoside binding is not controlled by the homotropic allosteric effect. We analyze structural differences between the HA-(sialoside)3 complex and isolated HA and evaluate the multiple-sialoside binding in the HA-sialoside complex. * Corresponding author. Fax: (+81)29-861-3171. E-mail: sawada-t@ aist.go.jp. † AIST. ‡ JST. § Kyoto University.

10.1021/jp1068895  2010 American Chemical Society Published on Web 11/04/2010

Binding of Influenza A Virus Hemagglutinin

Figure 1. Geometry-optimized structure of X-31 HA in complex with Neu5AcR2-6Galβ (24 060 atoms). White, green, and blue ribbons: HA1domain.Purpleribbon:HA2domain.YellowCPK:Neu5AcR2-6Galβ. Color lines: Asn-linked glycan. (A) Side view. (B) Top view.

kcal · mol-1 · Å-1. The rmsd of the peptide backbone (C, CR, N) between the initial structure and its energy minimum structure is 0.36 Å. Figure 2A summarizes the main geometrical details of the inter- and intramolecular interactions between the amino acid residues and one Neu5AcR2-6Galβ in the energy minimum X-31 HA-(Neu5AcR2-6Galβ)3 complex, as reported in our earlier work.7 The validity of the structures was confirmed by performing classical MD simulations under isobaric-isothermal (NPT) conditions at 296 K, 1.0 atm, to the X-31 HA in complex with single, double, and triple sialosides Neu5AcR2-6Galβ14Glc (Figure 2B and SI, Figures S2-S3 in Appendix S1). The crystallographic structure of the isolated X-31 HA9 (PDB ID: 1HGF) was also energy-minimized by the same procedure to evaluate the conformational change of HA between the isolated X-31 HA and the X-31 HA-(Neu5AcR2-6Galβ)3 complex. The rmsd of the peptide backbone between the crystal structure and its energy minimum structure is 0.34 Å. According to constant-NPT MD simulation, the energy minimum structure

J. Phys. Chem. B, Vol. 114, No. 47, 2010 15701 well represents X-31 HA at pre-equilibrium (SI, Figure S4 in Appendix S1). To investigate the conformational change of HA induced by Neu5AcR2-6Galβ binding, we estimated various rmsd’s between the two energy minimum structures of the X-31 HA-(Neu5AcR2-6Galβ)3 complex and isolated HA by utilizing VMD software14 (SI, Table S1 and Figure S5). The conformational change was also evaluated by comparing the pre-equilibrated complexes with isolated HA (SI, Figure S6). The classical MD simulations were carried out by the following procedure. The energy-minimized structures of X-31 HA in complex with three Neu5AcR2-6Galβ1-4Glc receptors involving crystallographically determined water were neutralized with sodium cations and immersed in a TIP3P15 water box with the thickness of 25.0 Å around the solute surface, as placed by AmberTools.16 Thus we obtained the solvated X-31 HA(Neu5AcR2-6Galβ1-4Glc)3 system (solute, about 24 000 atoms; TIP3P water, about 120 000 molecules). The corresponding systems with single and double Neu5AcR26Galβ1-4Glc were prepared by simply removing extra sialotrisaccharide from the energy minimum X-31 HA(Neu5AcR2-6Galβ1-4Glc)3 complex, followed by neutralization and hydration. The isolated X-31 HA system was also prepared with the same procedure on the basis of the energyminimized isolated X-31 HA. Classical MD simulations of these systems were performed by employing NAMD2.617 with PARM99 parameters,18 phi psi torsions by Duan et al.,19 and amino94 charges18 to the HA peptide, GLYCAM06e,20 for Neu5AcR2-6Galβ1-4Glc. Initial geometries of the systems were energy-minimized by the conjugate gradient method, heated gradually to 296 K under 1.01325 bar (1.0 atm) for 50 ps to relax TIP3P waters, maintained at 296 K, 1.0 atm, for 50 ps, and cooled gradually to 20 K under 1.0 atm for 50 ps to relax the whole system, and finally the whole systems were fully energy-minimized. After this, the whole systems were gradually heated to 296 K under 1.0 atm for 50 ps and pre-equilibrated at 296 K, 1.0 atm, for 200 ps. The pre-equilibration was carried out under isobaricisothermal (NPT) conditions using Langevin dynamics with Nose´-Hoover Langevin piston pressure control21,22 to 1.0 atm at 296 K. The SHAKE algorithm23 was employed to restrain bond length involving hydrogen atoms, and the SETTLE24 algorithm was used for TIP3P rigid water with the default tolerance 10-8 and the integration time step 1.0 fs with the velocity Verlet integration method25 (saving a snapshot every 5 ps), using periodic boundary conditions (cubic) and electrostatic interaction computed by the particle mesh Ewald method (PME)26 with the PME direct space tolerance 10-6 (default) and grid spacing 1.0 Å. The cutoff distance for van der Waals interactions and the short-range component of PME electrostatic was 12.0 Å (using no switching functions), and the time step between full nonbonded evaluations was 1.0 fs (fullElectFrequency and nonbondedFreq are 1.0 fs). 1,4 scaling factors of van der Waals and electrostatic interactions were 2.0 and 0.833333 (scnb ) 2.0, scee ) 1.2), and the dielectric constant was 1.0. On the basis of the energy minimum X-31 HA(Neu5AcR2-6Galβ)3 complex, we analyzed the multiple sialoside binding to the HA-sialoside complex (containing about 24 000 atoms) at the quantum-chemical level by employing the fragment molecular orbital method (FMO)27-29 with the polarizable continuum model (PCM)30,31 at the second-order MøllerPlesset perturbation theory (MP2) with the 6-31G(d) basis set. The FMO is a quantum-mechanical method based on fragmenting the system and performing ab initio calculations

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of fragments and their pairs in the electrostatic field of the whole system. FMO has been applied to a number of large systems, for example, SN2 reaction in aqueous solution,32 oligosaccharide in solution,33 silicon nanowire,34 red fluorescent proteins,35 prion protein,36 the photosynthetic reaction center,37 and protein-ligand complexes.38 There are many other fragment-based approaches: the electrostatically embedded many-body method,39 the kernel energy method,40 the elongation method,41 divide-and-conquer methods,42,43 and many others.44-49 FMO has been applied to influenza virus proteins in the gas phase,6,7,50-53 and to the best of our knowledge, this work reports the largest solvated PCM calculation (about 24 000 atoms) so far with a quantum-chemical method. The binding free energies ∆GPCM between the X-31 HA and Neu5AcR2-6Galβ are evaluated as

GPCM ) Ginternal + Ges + Gcav + Gdisp + Grep

Sawada et al.

Ginternal ) Egas + Gpold AB A B ∆GPCM ) GPCM - GPCM - GPCM

where the free energies GPCM are computed at 298 K at the FMO2-MP2/PCM[1(2)]/6-31G(d) level and B in the above equation for ∆GPCM is always sialoside (a-receptor in Figure 3, yellow CPK model), whereas A can be X-31 HA (Figure 3, complex A) or some complex of X-31 HA with sialoside (Figure 3, complexes B-D). ∆GPCM is the binding energy per single Neu5AcR2-6Galβ. GPCM consists of the following components. The solute internal energy Ginternal is given by a sum of the solute gas-phase energy Egas and the destabilization component Gpold of solvent-induced solute polarization; Ges is the solute-solvent electrostatic interaction energy (Ges includes the stabilization component of the solvent-induced polarization of the solute, which is typically equal54,55 to about -2Gpold); and Gcav is the cavitation energy

Figure 2. Geometrical information of the interactions between Neu5AcR2-6Galβ and active site amino acid residues in the X-31 HA complex. ( A) Inter- and intramolecular interactions in one active site of the energy minimum X-31 HA-(Neu5AcR2-6Galβ)3 complex, optimized with class II force field.7 (B) The corresponding information in the same complex at pre-equilibrium during 200 ps constant-NPT MD simulation. See SI, Figure S2 in Appendix S1, for more information.

Binding of Influenza A Virus Hemagglutinin

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Figure 3. Binding of three Neu5AcR2-6Galβ to trivalent X-31 HA. White, green, and blue ribbons: HA1 domain. Purple ribbon: HA2 domain. Color lines: Asn-linked glycan. Yellow CPK models represent a consequent Neu5AcR2-6Galβ (a-receptor) bound to the complex with some Neu5AcR2-6Galβ (b,c-receptors), shown as red CPK models. (A) HA + a-receptor. (B) HA/b-receptor complex + a-receptor. The distance between a- and b-receptors in terms of centroids of mass is about 42 Å (white dashed line). (C) HA/c-receptor complex + a-receptor. The distance between a- and c-receptors is about 42 Å. (D) HA/b,c-receptors complex + a-receptor.

(describing the loss of the solvent-free energy necessary to create a cavity for the solute). Gdisp is the solute-solvent dispersion interaction energy, and Grep is the solute-solvent exchangerepulsion interaction energy (i.e., the nonelectrostatic part of the interaction excluding the dispersion). The desolvation free energy ∆Gsolvation in complex formation is given by ∆Gsolvation ) ∆Gpold + ∆Ges + ∆Gcav + ∆Gdisp + ∆Grep, where each component such as ∆Ges is computed as the difference between the complex and isolated systems. The ∆Gsolvation shows the change in the solvation energy during the complex formation,55 when some surface of the interacting systems is desolvated. We retain the solvation label in the energy component symbol because it represents the effects of solvation, which in this particular case of complex formation corresponds to partial desolvation effects in solution, in other words, the difference of two solvation energies becomes a partial desolvation energy. The accuracy of FMO/PCM was evaluated on a representative set of polypeptides and proteins, and the solvation energies were shown to be accurate within 1 kcal/mol.30 The adequacy of FMO-MP2/PCM in describing native conformations of proteins has been shown.56 Further computational FMO details and fragmentation of the HA-sialoside complex are provided in SI Appendix S2 and Figure S7. All FMO calculations were performed with GAMESS.57 Results and Discussion We found that the structure of hemagglutinin changes very little upon complexation with three sialosides, as shown by the small rmsd values in the energy minimum structures (SI, Table S1). At the sialoside binding site, minor conformational changes of amino acid side chains were only observed in eight residues on the energy minimum structure (SI, Figure S5), and in comparison to the pre-equilibrium structures of X-31 HA in complex with single, double, and triple sialosides, isolated X-31 HA had no significant structural changes of the HA peptide backbone (SI, Figure S6). Therefore, we suggest that the sialoside binding to trivalent HA does not induce any conformational change of HA toward additional sialoside binding. However, very little conformational change is not a sufficient condition for no allosteric effect, and this motivates us to carry out a detailed energetic analysis of the trivalent X-31 HA-sialoside complex formation at the MP2 level of theory. On the basis of the above result, starting from the experimental structures, we analyzed the energy minimum geometries of X-31 HA in complex with three sialosides for the calculations of the system containing 1-2 sialosides as well, by removing extra molecules. The structures are shown in Figure 3, for complexes A, B, C, and D, containing 1, 2, 2, and 3 sialosides, respectively.

TABLE 1: Components of the Binding of X-31 Hemagglutinin and Neu5Acr2-6Galβ Sialosides at the FMO2-MP2/PCM[1(2)]/6-31G(d) Level energies relative to the binding in complex Aa entry energy, kcal/mol

complex A (1)b

complex B (2)b

complex C (2)b

complex D (3)b

-135.4 -44.0 -179.5

7.7 -0.3 7.4

7.8 -0.2 7.6

15.5 -0.5 15.0

1 2 3

∆Egas ∆Gpold ∆Ginternalc

4 5 6 7

∆Ges ∆Gcav ∆Gdisp ∆Grep

122.7 -3.1 54.6 -13.7

-7.4 0.0 0.0 0.0

-7.6 0.0 0.0 0.0

-15.0 0.0 0.0 0.0

8

∆Gsolvationd

116.5

-7.7

-7.8

-15.5

9

∆GPCMe

-18.9

0.0

0.0

0.0

a

Negative values mean a binding stronger than in complex A. The complexes are depicted in Figure 3; the number of sialosides is shown in parentheses. c Internal solute energy, sum of entries 1 and 2. d Desolvation energy, sum of entries 2 and 4-7. e Total energy, sum of entries 1 and 8. b

The weak trivalent X-31 HA-monovalent sialoside association with the intrinsic dissociation constant (KD) on the order5 of 10-3 M is achieved by the balance between the fast association rate (104-6 M-1 s-1) and fast dissociation rate (100-2 s-1) in the equilibrium solution.58,59 Because of the short lifetime of the HA-sialoside complex, the dynamic fluctuation of the trivalent HA structure induced by the sialoside binding should be small. Thus, the conformational and vibrational entropic change for the trivalent HA-monovalent sialoside association does not promote or obstruct an additional sialoside binding to the HA-sialoside complex. In other words, this allows us to study the relative energies of binding consequent sialosides to determine the homotropic allosteric effect. Although the solute entropic changes not explicitly considered in our work may be important to the absolute binding free energies, their influence should be small for the allosteric effect. First, we discuss the energetic property of binding between trivalent X-31 HA and a single sialoside at the FMO2-MP2/ PCM[1(2)]/6-31G(d) level, complex A (Table 1). The gas-phase binding energy ∆Egas is very exothermic (entry 1), as appears to be typical.55 To this, solvent adds polarization of the solute, which in PCM is divided into the follow two contributions:29 destabilizing (Gpold), related to how the internal energy of the solute is shifted up by solvation, and stabilizing, which is

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included in the solvent-solute electrostatic interaction energy Ges. In general, the complex formation decreases the absolute values of all solvent-related energy components because the solvent available surface is reduced. In other words, the ∆-components (∆Ges etc., Table 1) have the opposite sign compared to the absolute values (for separate systems). This is why the ∆Gpold is negative, ∆Ges is positive, etc., resulting in a positive desolvation energy ∆Gsolvation. Next, we look at the effect of adding more sialosides to the trivalent X-31 HA-sialoside complex. As a general remark, complexes B and C differ only in the structural chirality (Figure 3); because of the C3 symmetry, it makes a small difference if the second sialoside is added on the left- or right-hand side from the first sialoside. Indeed, the energy components for complexes B and C are very similar. Complex D, on the other hand, can be seen as combining the binding effects of both B and C, and the actual values in Table 1 appear to be additive (although they were calculated without this assumption). For example, placing the second sialoside to either site (complexes B and C) reduces the binding energy ∆Egas of the first sialoside by 7.7 and 7.8 kcal/mol, respectively (entry 1). Thus, when both sialosides are added (complex D), the resultant energy decrease is 15.5 kcal/mol. The ∆GPCM analysis of X-31 HA-Neu5AcR2-6Galβ complexes implies that additional sialosides have no overall effect on the HA-sialoside binding (complexes B-D in entry 9, Table 1), i.e., no homotropic allosteric effect. However, individual components show a considerable change. For instance, ∆Egas for complexes B and C tells us the corresponding contribution to the binding of the second sialoside is positive (7.7 or 7.8 kcal/mol), which means that the first sialoside reduces the HA affinity to the consequent sialoside in the gas phase. This is rather interesting, as the distance between the active sites is about 42 Å (Figure 3), and this very long-range effect can be seen to be electrostatic in nature, originating from the negative charge on sialoside with the total charge of -21 on X-31 HA. What is even more interesting is the fact that this affinity reduction is perfectly canceled by the affinity increase due to the solute-solvent electrostatics ∆Ges (entry 4) and the rather insignificant polarization term ∆Gpold (entry 2). Thus, the presence of the first sialoside enhances the electrostatic interaction between the solvent-representing charges and the consequent sialoside, which again shows how far reaching electrostatic effects are. The other terms, the changes in the cavitation energy ∆Gcav, the solvent-solute exchangerepulsion energy ∆Grep, and the solvent-solute dispersion energy ∆Gdisp are nearly zero (entries 5-7) because they are relatively short-ranged determined by the cavity and its surface, and some change at one site has no effect on the other. As a remark, it is clear that the solvent effects are absolutely crucial for the studies of the sialoside binding, as the conclusions would be based only on the trends for ∆Egas without the ∆Gsolvation term, and the prediction would be very different; namely, there is a considerable negative homotropic allosteric effect (decreasing the affinity with consequent ligands). Experimentally, the weak HA-sialoside monovalent association is enhanced by the polyvalent interaction effect. Indeed, the interaction between the soluble X-31 HA rosettes and the fetuin-bound plate had a 104-fold lower apparent KD measured by surface plasmon resonance analysis under the homogeneous binding model, where fetuin was a sialoglycoprotein containing three Asn-linked sialoglycans.60 Besides, the apparent KD in the Aichi/2/1968 virion-gangliosides-contained lipid layer interaction was 107-fold lower than the KD in the monovalent system.61

Sawada et al. However, these polyvalency-induced affinities are not caused by a positive sialoside homotropic allosteric effect but appear because of the small loss of the translational and rotational entropies in reassociation between released-HA and the substrate containing multiple sialoside receptors on its surface. In other words, mass transport limitation is caused by nonequilibration between the HA-sialoside encounter phase and the bulk phase.62 The implications of the elucidated binding to drug design are as follows. Because the polyvalent effect is not observed in the virion-monovalent Neu5AcR2-6Galβ1-4Glc system with millimolar order of KD,63,64 the HA-sialoside association can be controlled by a design of polyvalent sialoside derivatives such as the sialoglycolipid-buried membrane,65 soluble sialoglycoconjugates,66,67 and the sialoside-bound plate.68-70 However, these conjugates are not suitable to be used as a drug due to their large molecular sizes. In essence, to inhibit the HA-sialoside association, one should control chemically the HA-inhibitor encounter phase to achieve a small KD on the HA-inhibitor complex, regardless of its monovalency or polyvalency. One of the approaches without sialoglycoconjugates is a selective chemical ligation71-73 between HA and the sialoside derivative. The sialoside derivative requires a sialoside moiety with a reactive group to form a selective covalent bond with the amino acid residue side chains (nucleophile) in HA under mild conditions and a suitable linker between the sialoside moiety and the reactive group. In this approach, a small KD on the intramolecular HA-sialoside moiety (inhibitor) complex can be obtained by a very fast association rate and moderate dissociation rate on the intramolecular complex. In contrast, the small KD in the HA-polyvalent sialoside system originates in moderate association rate and very slow dissociation rate on the intermolecular HA-sialoside complex. In summary, we have demonstrated that the HA-sialoside binding is not regulated by the sialoside homotropic allosteric effect, as shown by the FMO2-MP2/PCM[1(2)]/6-31G(d) calculations. The energy minimum geometries of HA in the isolated and bound states are very similar. These results suggest the simple binding mode in the soluble trivalent HA-monovalent sialoside interaction under equilibrium solution, and this information can be utilized for design of selective HA-sialoside association inhibitors, providing potential means to cope with influenza pandemics. Acknowledgment. This work was supported by contract grant sponsors: CREST (JST, Japan) and The Next Generation Super Computing Project and Nanoscience Program (MEXT, Japan). Supporting Information Available: Definitions for the dihedral angles of the R2-6 bond on Neu5AcR2-6Galβ (Figure S1), isobaric-isothermal (NPT) MD simulations of the X-31 HA-(Neu5AcR2-6Galβ1-4Glc)3 system, the corresponding systems with single and double sialosides, and isolated X-31 HA (Appendix S1 and Figures S2-S4); structural alignment and rmsd in the energy minimum structures between the X-31 HA-(Neu5AcR2-6Galβ)3 complex and isolated X-31 HA (Table S1); minor conformational changes of amino acid side chains at the sialoside binding site in the energy minimum structure (Figure S5); various rmsd’s of the pre-equilibrium X-31 HA in isolated and single, double, and triple Neu5AcR2-6Galβ1-4Glc complexes (Figure S6); and details of the FMO computation (Appendix S2 and Figure S7). This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Horimoto, T.; Kawaoka, Y. Nat. ReV. Microbiol. 2005, 3, 591. (2) Skehel, J. J.; Wiley, D. C. Annu. ReV. Biochem. 2000, 69, 531.

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