Insights into the binding specificity of wild type and mutated wheat germ agglutinin towards Neu5Acα(2-3)Gal: a study by in silico mutations and molecular dynamics simulations.

Research Article Received: 23 October 2013,

Revised: 2 February 2014,

Accepted: 2 February 2014,

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI: 10.1002/jmr.2369

Insights into the binding specificity of wild type and mutated wheat germ agglutinin towards Neu5Acα(2-3)Gal: a study by in silico mutations and molecular dynamics simulations Ponnusamy Parasuramana, Veeramani Murugana, Jeyasigamani F. A. Selvina, M. Michael Gromihab, Kazuhiko Fukuic and Kasinadar Velurajad* Wheat germ agglutinin (WGA) is a plant lectin, which specifically recognizes the sugars NeuNAc and GlcNAc. Mutated WGA with enhanced binding specificity can be used as biomarkers for cancer. In silico mutations are performed at the active site of WGA to enhance the binding specificity towards sialylglycans, and molecular dynamics simulations of 20 ns are carried out for wild type and mutated WGAs (WGA1, WGA2, and WGA3) in complex with sialylgalactose to examine the change in binding specificity. MD simulations reveal the change in binding specificity of wild type and mutated WGAs towards sialylgalactose and bound conformational flexibility of sialylgalactose. The mutated polar amino acid residues Asn114 (S114N), Lys118 (G118K), and Arg118 (G118R) make direct and water mediated hydrogen bonds and hydrophobic interactions with sialylgalactose. An analysis of possible hydrogen bonds, hydrophobic interactions, total pair wise interaction energy between active site residues and sialylgalactose and MM-PBSA free energy calculation reveals the plausible binding modes and the role of water in stabilizing different binding modes. An interesting observation is that the binding specificity of mutated WGAs (cyborg lectin) towards sialylgalactose is found to be higher in double point mutation (WGA3). One of the substituted residues Arg118 plays a crucial role in sugar binding. Based on the interactions and energy calculations, it is concluded that the order of binding specificity of WGAs towards sialylgalactose is WGA3 > WGA1 > WGA2 > WGA. On comparing with the wild type, double point mutated WGA (WGA3) exhibits increased specificity towards sialylgalactose, and thus, it can be effectively used in targeted drug delivery and as biological cell marker in cancer therapeutics. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: cyborg lectin; wheat germ agglutinin; sialylglycans; molecular modeling; molecular dynamics; binding specificity; binding free energy; cell marker

INTRODUCTION

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Lectins are sugar-binding proteins of non-immune origin having unique recognition to diverse carbohydrate structures, which mediate numerous biological processes such as cell–cell and host–pathogen interactions, regulating cell adhesion and cancer metastasis (Goldstein et al., 1980; Hart, 1980; Sharon and Lis, 1989; Lis and Sharon, 1998; Vijayan and Chandra, 1999; Sharon and Lis, 2004). They originate from plants, animals, bacteria, and viruses, and their isolated complex structures are reported earlier (http://www.cermav.cnrs.fr/databank/lectine; Damodaran et al., 2008). WGA is a plant lectin, which specifically recognizes sialyloligosaccharides found on the cell surfaces that serve as receptors for viruses, bacteria, lectins, toxins, mycoplasma, and protozoa (Varghese et al., 1992; Varki, 1992; Kelm and Schauer, 1997; Varki, 1997). Plant lectins are non-enzymatic in nature and can be easily purified and characterized (Etzler, 1985; Pusztai, 1991; Chandra et al., 2006; Peter et al., 2009; Liu et al., 2010). Plant lectin has the potential to agglutinate the blood cells exhibiting affinity towards specific carbohydrates, thereby discriminating different blood groups (Yim et al., 2001). WGA is found as a highly stable non-covalent dimer having 16 disulfide bonds, two independent binding sites per subunit that

J. Mol. Recognit. 2014; 27: 482–492

specifically bind to two different saccharides GlcNAc and NeuNAc [Protein Data Bank (PDB) code: 1WGC] (Wright, 1990; Lis and Sharon, 1991). WGA acts as antimicrobial agents in drug

* Correspondence to: Kasinadar Veluraja, Department of Physics, Noorul Islam University, Kumaracoil, Thuckalay, Kanyakumari 629 180, Tamilnadu, India. E-mail: [email protected] a P. Parasuraman, V. Murugan, J. F. A. Selvin Department of Physics, Manonmaniam Sundaranar University, Tirunelveli 627 012, India b M. M. Gromiha Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600 036, India c K. Fukui National Institute of Advanced Industrial Science and Technology (AIST), Molecular Profiling Research Center for Drug Discovery (molprof), Tokyo 135-0064, Japan d K. Veluraja Department of Physics, Noorul Islam University, Kanyakumari 629 180, India Abbreviations used: WGA, wheat germ agglutinin; Neu5Ac, N-Acetylneuraminic acid (sialic acid); Gal, galactose; Glc, glucose; MD, molecular dynamics; BM, binding mode; MM-PBSA, molecular mechanics Poisson–Boltzmann surface area.

Copyright © 2014 John Wiley & Sons, Ltd.

BINDING SPECIFICITY OF MUTATED WGAS TOWARDS SIALYLGLYCANS

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interact with binding site residues, and no water molecules are observed in their proximity. In the present study, in silico mutations are performed at the binding site of WGA, because mutating the active site residues alter the binding specificity of WGA towards saccharide binding (Sharon and Lis, 1995). Three different in silico mutations are carried out at the binding site of WGA (Figure 1(b)) in order to examine the change in the binding specificity of cyborg lectins (mutated lectins) towards the sialylgalactose [Neu5Acα(2-3)Gal – N23G]. Twenty nanoseconds MD simulation studies are performed on wild type and mutated WGAs in complex with N23G to investigate the bound conformational flexibility of N23G in the binding pocket of WGAs and the binding specificity of mutated WGAs with sialylgalactose complex.

MATERIALS AND METHODS Starting structure The three dimensional coordinates of wheat germ agglutinin are taken from the PDB (PDB code: 1WGC) (Wright, 1990; Berman et al., 2000). Subunit A of 1WGC is taken for in silico mutation study because the two identical well-separated subunits A and B are independent and the active site residues are conserved. The conformation of N23G is kept at [ΦN23G, ΨN23G: 69°, 9°] in the starting structure for the four complexes (WGA, WGA1, WGA2, and WGA3). The schematic representation of the N23G with glycosidic torsional angles marked is shown in Figure 2. Amino acid mutation The amino acid residues are mutated using in-house developed FORTRAN programs (Intel Corporation, USA; Version: Intel Fortran Compiler 9.1 for Linux). The mutations are rationalized by the idea

Figure 1. (a) Hydrogen bonding interactions in the crystal structure of 1WGC (subunit A) and (b) native sequences of wheat germ agglutinin are green in color and the mutated residues are red, blue, and magenta in color for WGA1 (S114N), WGA2 (S114N and G118K), and WGA3 (S114N and G118R), respectively.


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delivery and induces apoptosis of tumor cells (Ogawara et al., 1985; Kim et al., 1993; Wirth et al., 1998; Wirth et al., 2002; Bies et al., 2004; Gastman et al., 2004; Mo and Lim, 2005; Clark and Mao, 2012). α2,3-Linked and α2,6-linked sialylation is identified to be prominent in prostate cancer (Peracaula et al., 2003; Meany et al., 2009; Kim and Misek, 2011). Many experimental observations that include isolation, crystallization, purification, composition, and binding specificity of WGA are reported (Nagata and Burger, 1972; Allen et al., 1973; Nagata and Burger, 1974; Wright, 1992). X-ray crystallographic and NMR studies are employed in studying the WGA binding with sugars (Wright, 1974; Wright et al., 1974; Wright, 1977; Kronis and Carver, 1982; Wright, 1987). Apart from disulphide bridges and hydrophobic interactions, hydrogen bonds play a vital role in dictating the specificity of WGA towards saccharides (Sharon and Lis, 1995; Sharma et al., 2009). The binding specificity of lectins could be changed by means of substitutions of one or two amino acid residues in the binding site (Sharon and Lis, 1995). Also, mutations at specific amino acid residues may lead to a change in shape of the binding pocket (Mishra et al., 2012). Single amino acid substitutions in the hemagglutinins that can change the receptor binding specificity were reported by Rogers et al. (1983). Mutations by using polymerase chain reaction (Higuchi et al., 1988) are carried out to examine the amino acid residues essential for ligand binding (Adar and Sharon, 1996) and changes in the substrate specificity upon mutations (Hardt and Laine, 2004). Cyborg lectins of Bauhinia purpurea lectin are designed by mutating the cDNA coding domains (Yamamoto et al., 2000). Best BMs and their relative energies are reported for in silico-mutated bacterial lectin by Adam et al. (2008). The mutated WGA with increased specificity towards sialylglycans can be effectively used as biological cell marker in cancer therapeutics (Wright, 1990; Lis and Sharon, 1991; Peracaula et al., 2003; Meany et al., 2009; Kim and Misek, 2011; Clark and Mao, 2012). Molecular dynamics simulation is used in studying the conformational and dynamical properties of glycans and protein–carbohydrate interactions (Tsujishita et al., 1997; Karplus and McCammon, 2002; Fadda and Woods, 2010) and the internal dynamics of folded proteins (McCammon et al., 1977; Daggett, 2002). MD simulations of sialylglycans and their conformational preferences, interactions, and role of water in structural stabilization have been reported by Veluraja and co-workers (Xavier Suresh and Veluraja, 2003; Selvin et al., 2012; Venkateshwari and Veluraja, 2012a, 2012b), and the binding specificity of sialylglycans in complex with hemagglutinins of influenza A virus and SelectinE has been deduced as well (Veluraja and Seethalakshmi, 2008; Priyadarzini et al., 2012). Hydrogen bonds and water bridges that are involved in structural stabilization of lectin–carbohydrate complexes using MD simulation are reported (Bryce et al., 2001; Clarke et al., 2001; Kadirvelraj et al., 2008; Sharma et al., 2009; Nurisso et al., 2010; Fadda and Woods, 2011). The dissociation pathway of WGA and the intramolecular motion of sugars bound with WGA have been investigated using MD simulations (Neurohr et al., 1980; Tagami et al., 2009). In the crystal structure of WGA-2,3 sialyllactose complex (1WGC), only two residues (Ser114 and Glu115) interact with the sialyllactose, and two hydrogen bonds are observed between (i) OG of Ser114 and carboxylate oxygen of Neu5Ac and (ii) OE2 of Glu115 and acetamide nitrogen of Neu5Ac (Figure 1(a)). Galactose and glucose of 2,3 sialyllactose do not

P. PARASURAMAN ET AL.

Figure 2. The schematic representation of Neu5Acα(2-3)Gal [ΦN23G = C1C2-O2-C3; ΨN23G = C2-O2-C3-H3; R1 = NHCOCH3; R2 = (CHOH)2CH2OH].

of enhancing the interactions between the sialylgalactose and binding site residues of lectin. The amino acid mutants are chosen based on the length of the side chains that can likely to have better interactions with the sugar. The first mutation is a single point mutation in which Ser114 is substituted by Asn114 (WGA1); the second mutation is a double point mutation in which Ser114 and Gly118 are substituted by Asn114 and Lys118 (WGA2); and the third mutation is also a double point mutation in which Ser114 and Gly118 are substituted by Asn114 and Arg118 (WGA3) as shown in Figure 1(b). The stability changes (ΔΔG) of mutated WGAs have been calculated after the substitution of mutants Asn114, Lys118, and Arg118 using the CUPSAT server (Cologne University Protein Stability Analysis Tool; developed by Dr. Vijaya Parthiban; Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK. Email: [email protected]). The calculated ΔΔG values represent the protein stability changes upon amino acid mutation. This stability change is calculated from atom potentials and dihedral angle distribution. The predicted negative and positive ΔΔG values mean the destabilizing and stabilizing effect, respectively. The ΔΔG values of the aforementioned mutated residues are 0.65, 2.92, and 3.24 kcal/mol, respectively (Topham et al., 1997; Gromiha et al., 1999; Parthiban et al., 2006; Parthiban et al., 2007a, 2007b). Molecular Dynamics simulation

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The input files for simulations are prepared using General AMBER Force Field (GAFF) for saccharide and ff99 for protein of AMBER9 (Wang et al., 2000; Wang et al., 2004; Case et al., 2006). GAFF force field for carbohydrates produces reliable results when used alongside ff99 for proteins in protein–carbohydrate complex simulations (Veluraja and Margulis, 2005; Yan et al., 2008; Priyadarzini et al., 2012), and the charge method used in GAFF is HF/6-31G* restrained electrostatic potential charge. Necessary chlorine ions are added to neutralize the complex. Particle mesh Ewald is used to treat the long-range electrostatic interactions (Darden et al., 1993). The complex system that includes protein, sialylgalactose, and the ions is solvated using TIP3P water (Jorgensen et al., 1983). The solvated system is equilibrated for 2000 steps and then simulated for 20 ns duration using NAnoscale Molecular Dynamics (NAMD) (Phillips et al., 2005). During the production simulations, the number of particles, pressure, and temperature of the system are kept constant at 1 atm. To mimic the biological environment, temperature is maintained at 300 K throughout the simulations. The MD trajectories are recorded for every picosecond, and 20 000 structures are collected for a typical 20 ns simulation. The trajectories are analyzed using visual molecular dynamics (Humphrey et al., 1996). Total pair wise interaction energy between the interacting active site residues and


sialylgalactose has been calculated using NAMD, and the plausible BMs are predicted. The binding free energy calculations have been carried out by subtracting the free energies of the unbound protein and carbohydrates from the free energy of the bound complex as ΔGbind = ΔGcomplex – (ΔGprotein + ΔGsugar) using the MM-PBSA script of AmberTools13 ((Assisted Model Building with Energy Refinement); developed by David A. Case, The Scripps Research Institute, USA) (Srinivasan et al., 1998; Bryce et al., 2001; Gilson and Zhou, 2007; Miller et al., 2012). Atomic visualization of wheat germ agglutinin–sialylgalactose interactions is rendered by using UCSF Chimera (Pettersen et al., 2004) and MolScript (Kraulis, 1991).

RESULTS AND DISCUSSION Conformational flexibility of N23G at the binding pocket of wheat germ agglutinins The glycosidic torsions of N23G observed from 20 ns MD simulations of WGA, WGA1, WGA2, and WGA3 complexes are depicted (Figure 3). Figure 3 shows that N23G prefers three distinct conformations viz A, B, and C and the respective (ΦN23G, ΨN23G) are ( 150°, 30°), ( 100°, 50°), and ( 70°, 10°). The population propensities of N23G at the binding pocket of native WGA are similar to the propensities in free state (Veluraja and Margulis, 2005; Veluraja et al., 2010; Selvin et al., 2012). However, difference in propensities of conformations is observed when N23G is at the binding pocket of cyborg lectins (WGA1, WGA2, and WGA3). N23G exists in ( 69°, 9°) conformation in the crystal structure (Wright, 1990), which is also one of the conformations observed from our MD simulations. The MD trajectories obtained from four simulations and the corresponding pair wise interaction energy plots are analyzed for possible hydrogen bonds (distance between two electronegative atoms X and Y is 2.6 to 3.2 Å and the angle ∠H-Χ…Y ≤ 30˚), residence time of water that mediates the hydrogen bonds, hydrophobic interactions, and the movement and relative orientation of sialylgalactose inside the binding pocket of WGAs. The starting structure for the simulation exhibits sialylgalactose in a single orientation. During dynamics, the sialylgalactose moves inside the binding pocket and interacts with the active site residues. When there is a change in relative orientation and conformation of N23G, the interaction pattern changes, which includes the change in sets of hydrogen bonds and the hydrophobic effects. Based on the analysis, different BMs are proposed for N23G binding with WGAs. The binding free energy calculations are carried out for different BMs, and the results substantiate the proposed BMs. The BM with the global minimum energy is denoted as BM1, and the relatively higher energies are denoted as BM2 and BM3. Molecular Dynamics simulation of WGA–N23G complex Molecular dynamics simulation of 20 ns duration is performed for the complex WGA–N23G. The pair wise interaction energy between N23G and the interacting active site residues Leu112, Gly113, Ser114, Glu115, Gly118, and Gly119 is calculated and plotted as Figure 4(a). Based on the analysis of MD trajectories and the pair interaction energy, it is proposed that WGA can accommodate N23G in three different BMs, which are denoted as BM1 (11 800–16 800 ps), BM2 (7900–11 800 ps), and BM3 (2500–7900 ps). BM1 has the global minimum energy. BM2 and BM3 have relatively 9.2 and 19.2 kcal/mol energy higher than BM1. The direct and water mediated hydrogen bonds that stabilize each BM and the residence time of water involved in water mediated hydrogen bonds are given in Table 1, and the


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Figure 3. Glycosidic torsional maps for N23G in the complexes of (a) N23G–WGA, (b) N23G–WGA1, (c) N23G–WGA2, and (d) N23G–WGA3.

Figure 4. Total pair wise interaction energy between N23G and the interacting active site residues of (a) WGA–N23G, (b) WGA1–N23G, (c) WGA2–N23G, and (d) WGA3–N23G complexes.

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of Leu112. Acetamide nitrogen of Neu5Ac makes water mediated hydrogen bond with amide nitrogen of Glu115. O8 of Neu5Ac makes both direct and water mediated hydrogen bonds with OE1/OE2 of Glu115 and water mediated hydrogen bond with OG of Ser114. O4 of galactose forms water mediated hydrogen bond with amide nitrogen of Ser114. A total



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relative energies are given in Table 2. When N23G exists in BM1, carboxylate oxygen of Neu5Ac forms direct hydrogen bonds with amide nitrogen and OG of Ser114 in addition to the water mediated hydrogen bond with amide nitrogen of Glu115. Another carboxylate oxygen forms water mediated hydrogen bonds with amide nitrogen of Ser114 and carboxylate oxygen

486

N23G

Receptor


Binding mode2 (BM2)

Binding mode1 (BM1)

Binding modes


O1B O2B

O8 O9

N5

O4

O1D

O1S

O6B

O4B

O2B

O10 O1B

O9

N5 O7 O8

O4

O1D

O1S

Receptor atom

— — — — OG-Ser114 — — — — — WAT-N-Ser114 N-Glu115 N-Ser114 WAT-OE1 Glu115 WAT-OE1 Glu115

OG-Ser114 N-Ser114 WAT-N-Glu115 WAT-O-Leu112 WAT-N-Ser114 — — WAT-N-Glu115 — WAT/OE1-Glu115 WAT-OE2-Glu115 WAT-OG-Ser114 — — — — — — — — — WAT-N-Ser114

Protein atoms in WGA

Protein atoms in WGA1 ND2-Asn114 — — WAT-N-Asn114 — N-Asn114 O-Leu112 WAT/O-Leu112 WAT-OG-Ser127 — — — — — O-Leu112 WAT1-O-Ser127 WAT2-O-Ser127 — — WAT-OG-Ser127 — WAT-OD1-Asn114 WAT-ND2-Asn114 — — WAT-N-Gly119 WAT-O-Asn114 WAT/O-Asn114 WAT-ND2-Asn114 — — WAT/ND2-Asn114 OD1-Asn114 — — — — —

Residence time of water in ps — — 8.7 6.5 7.9 — — 4.8 — 3.5 3.3 3.9 — — — — — — — — — 3.5 — — — — — — — — — — — 3.3 — — 5.5 3.4

— — — 7.9 — — — 3.6 3.4 — — — — — — 3.7 3.9 — — 6.9 — 3.5 3.9 — — 3.6 3.6 7.9 4.8 — — 4.9 — — — — — —

Residence time of water in ps WAT-OH-Tyr159 — — — — — — — WAT-NZ-Lys118 WAT-O-Cys126 WAT-O-Cys153 WAT-OH-Tyr159 N-Asp129 O-Ser127 — WAT1-NZ-Lys118 WAT2-NZ-Lys118 — — — — WAT-OD2-Asp129 — — — WAT-NZ-Lys118 — — — N-Asp129 N-Thr128 OH-Tyr159 — — — — — O-Thr128

Protein atoms in WGA2 6.7 — — — — — — — 7.6 4.4 4.9 5.8 — — — 6.8 4.7 — — — — 7.4 — — — 6.4 — — — — — — — — — — — —

Residence time of water in ps

Table 1. Interactions between the receptor N23G and the interacting binding site residues of WGA, WGA1, WGA2, and WGA3

WAT/NH2-Arg118 — — NH1-Arg118 — — — — — — — — — — — O-Asn114 O-Arg118 WAT/O-Glu115 WAT-N-Gln106 O-Arg118 N-Gly119 NH1-Arg118 ND2-Asn114 WAT-NE1-Trp107 WAT/OE2-GLU115 WAT-NH1-Arg118 ND2-Asn114 NH1-Arg118 WAT-NH2-Arg118 — — — — — NH2-Arg118 — — WAT-ND2-Asn114

Protein atoms in WGA3 9.8 — — — — — — — — — — — — — — — — 6.3 5.9 — — — — 7.9 9.9 5.3 — — 7.5 — — — — — — — — 6.1



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— — — — — — — — — — — — NH2-Arg118 — — — — — — — 5.8 7.7 — — — — — — — — WAT-O-Ser127 WAT-NZ-Lys118 — — — — — — — —

Molecular dynamics simulation of WGA1–N23G complex In WGA1–N23G complex, Ser114 is replaced by Asn114 (S114N) as a single point mutation. The pair wise interaction energy between N23G and the interacting residues Leu112, Asn114, Glu115, and Ser127 is calculated (Figure 4(b)). Two possible BMs BM1 and BM2 are proposed for this complex. In BM1 of WGA1-N23G complex, a total of 13 hydrogen bonds (5D, 8 W) stabilize the structure (Figure 6(a)). In addition to the hydrogen bonding interactions, a hydrophobic cluster is observed between Neu5Ac and Asn114, Cys117 which is a prominent interaction (Banerjee et al., 1996; Ambrosi et al., 2005) in stabilizing the complex (Figure 6(b)). Eight hydrogen bonds (3D, 5 W) stabilize BM2, and only two residues Asn114 and Gly119 interact with N23G (Table 1). Because of the change in orientation of N23G, the residues Leu112 and Ser127 lose their interactions, and Gly119 makes a water mediated hydrogen bond with N23G. The atomistic level interactions observed in the BMs BM1 and BM2 are given in Table 1. BM2 has 12.6 kcal/mol energy higher than BM1, and this energy difference can be accounted for the loss of hydrophobic interactions and the hydrogen bonds in BM2. MM-PBSA free energy calculations also propose that BM2 has 7.9 kcal/mol energy higher than BM1 (Table 2). Hence, it can be concluded that WGA1 shows better binding to N23G than the wild type.

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WGA, wheat germ agglutinin.

O10

N5 O8 Binding mode3 (BM3)

O4B O6B

— — — — — — 3.6 — — — — — — N-Gly119 O-Ser114 OG-Ser114 WAT-OE1-Glu115 OE2-Glu115 N-Ser114 N-Glu115

— — — — — — — — — —

— — — — — — — — — —

Residence time of water in ps Residence time of water in ps Receptor

Binding modes

of 11 hydrogen bonds [3D (Direct), 8 W (Water mediated)] are involved in the structural stabilization of BM1, and three amino acid residues Leu112, Ser114, and Glu115 are involved in the hydrogen bonding interactions (Table 2). The interaction diagram for BM1 of WGA–N23G complex is given in Figure 5. In BM2, three residues Ser114, Glu115, and Gly119 interact with N23G, and the total number of hydrogen bonds is 8 (5D, 3 W). Leu112 moves away from N23G, Gly119 comes in contact with N23G, and the corresponding energy shift can be observed in pair interaction energy at 11 800 ps. In BM3, the interactions are reduced to five (4D, 1 W) and only two amino acid residues Ser114 and Glu115 interact with N23G. Galactose totally loses its interactions with the protein in this BM (Table 1). Binding free energy calculations are carried out for the three BMs of WGA–N23G complex. The calculated binding free energy for BM1, BM2, and BM3 are 12.8, 6.4, and 3.8 kcal/mol, respectively (Table 2). In-depth analysis of the trajectories and the pair interaction energy plots using in-house developed FORTRAN program shows that the BMs are independent of the conformational propensity of N23G. Also, the mutation in the binding site residues does not alter the glycosidic torsional preferences.

Molecular Dynamics simulation of WGA2–N23G complex In WGA2, double point mutation is carried out by substituting Ser114 with Asn114 (S114N) and Gly118 with Lys118 (G118K). The pair wise interaction energy is calculated between N23G and the interacting residues Leu112, Asn114, Glu115, Lys118, Ser127, Thr128, Asp129, and Tyr159 (Figure 4(c)). Two modes of binding, BM1 and BM2, are observed for this complex. Analysis of the trajectories corresponding to BM1 reveals 12 hydrogen bonds (2D, 10 W) as shown in Figure 7(a). An elegant network is formed by the specific hydrogen bonds (Ochoa, 1981; Banerjee et al., 1996; Ambrosi et al., 2005) that start with



487

Table 1. (Continued)

Receptor atom

Protein atoms in WGA

Protein atoms in WGA1






488



7 (4D,3 W)

BM2

7 (4D,3 W)

BM2 15 (10D,5 W)

8 (3D,5 W) 12 (2D,10 W)

BM2 BM1

BM1

11 (3D,8 W)a 8 (5D,3 W) 5 (4D,1 W) 13 (5D,8 W)

Number of hydrogen bonds

BM1 BM2 BM3 BM1

Possible modes of binding

2

6

3 3 2 3 (hydrophobic interaction) 2 6 (network of hydrogen bonds) 5

Number of interacting residues

Asn114, Gly119 Lys118, Cys126, Ser127, Asp129, Cys153, Tyr159 Lys118, Ser127, Thr128, Asp129, Tyr159 Gln106, Trp107, Asn114, Glu115, Arg118, GLy119 Asn114, Arg118

Leu112, Ser114, Glu115 Ser114, Glu115, Gly119 Ser114, Glu115 Leu112, Asn114, Ser127

Interacting residues

20.3

0.0

14.0

12.6 0.0

0.0 9.2 19.2 0.0

Relative interaction energy (kcal/mol)

WGA, wheat germ agglutinin; MM-PBSA, Molecular Mechanics Poisson–Boltzmann Surface Area; BM, binding mode. a D, direct hydrogen bond; W, water mediated hydrogen bond. bEnergy in kilocalorie per mole.

WGA3 (S114N and G118R)

WGA2 (S114N and G118K)

WGA1 (S114N)

WGA

Wild type/ mutated WGAs

23.5 ± 4.8

37.3 ± 5.3

7.3 ± 5.3

15.3 ± 5.2 13.8 ± 5.0

12.8 ± 5.5 6.4 ± 4.9 3.8 ± 5.7 23.2 ± 4.7

Binding free energyb with standard deviation for the complexes using MM-PBSA

13.8 ± 4.8

0.0 ± 5.3

6.5 ± 5.3

7.9 ± 5.2 0.0 ± 5.0

0.0 ± 5.5 6.4 ± 4.9 9.0 ± 5.7 0.0 ± 4.7

Relative free energyb with standard deviation for the complexes using MM-PBSA

1

3

2

4

Order of binding specificity

Table 2. Possible number of binding modes, hydrogen bonds, hydrophobic interactions, and the binding free energy for the complexes of WGA, WGA1, WGA2, and WGA3 with N23G


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Figure 5. Atomic level interactions in BM1 of WGA–N23G complex.

carboxylate oxygen of Neu5Ac and ends with another carboxylate oxygen via O4 of galactose; OD2/OD1 of Asp129 and OH of Tyr159 is shown in Figure 7(b). In this network, observed water mediated hydrogen bond between O1 of Neu5Ac and O4 of galactose is similar to the one that is observed in the solution state dynamics of N23G, which stabilize the disaccharide at ( 150°, 30°) conformation (Veluraja and Rao, 1984; Veluraja and Margulis, 2005; Selvin et al., 2012). The atomistic level interactions between N23G and the interacting residues of WGA2 observed in BM1 and BM2 are given in Table 1. BM1 is relatively 14.0 kcal/ mol lower than BM2. The binding free energy calculations on the two different BMs of WGA2–N23G complexes show that BM1 is 6.5 kcal/mol energy lower than BM2 (Table 2). The binding of WGA2 is stronger than the binding of WGA but weaker than the binding of WGA1 to N23G.

Figure 6. (a) Direct and water mediated hydrogen bonds observed in BM1 of WGA1–N23G complex, and (b) hydrophobic cluster observed between Neu5Ac and Asn114, Cys117 in BM1.

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Figure 7. (a) Direct and water mediated hydrogen bonds observed in BM1 of WGA2–N23G complex, and (b) view of hydrogen bonding network between N23G and Asp129, Tyr159 observed in BM1.

P. PARASURAMAN ET AL. Molecular Dynamics simulation of WGA3—N23G complex In WGA3, Ser114 is substituted by Asn114 (S114N), and Gly118 is replaced by Arg118 (G118R). The total pair wise interaction energy plot between N23G and the interacting residues Leu112, Asn114, Glu115, Arg118, and Ser127 is shown in Figure 4(d). MD trajectories and pair interaction energy analysis reveal that two possible BMs BM1 and BM2 are observed for WGA3–N23G complex. N23G forms 15 hydrogen bonds (10D, 5 W) in BM1 (Table 2), and the interactions observed are described in the succeeding texts. Carboxylate oxygen of Neu5Ac forms direct hydrogen bond with NH1/NH2 of Arg118. This interaction is the key interaction of BM1 and BM2 and prevails up to 17.5 ns (Figure 9). This suggests that the mutant (substituted) amino acid residue Arg118 might be crucial for Neu5Ac binding. The anomeric oxygen of galactose forms direct hydrogen bond with carboxylate oxygen of Asn114, Glu115, Arg118, and water mediated hydrogen bonds with amide nitrogen and carboxylate oxygen of Gln106 and Glu115, respectively (Figure 8). The water mediated hydrogen bonds are the integral part of the structural stabilization of WGA3–N23G complex similar to the earlier reports (Banerjee et al., 1996; Kadirvelraj et al., 2008; Sharma et al., 2009; Nurisso et al., 2010; Fadda and Woods, 2011). O2 of galactose makes direct hydrogen bonds with carboxylate oxygen of Arg118 and amide nitrogen of Gly119. O4 of galactose makes direct hydrogen bonds with ND2 of Asn114 and NH1/NH2 of Arg118. O6 of galactose makes direct and water mediated hydrogen bonds with OE2 of Glu115 and water mediated hydrogen bond with NE1 of Trp107. A representative plot indicating these hydrogen bonds is given as Figure 8, and the atomistic level interactions between N23G and the binding site residues of WGA3 are given in Table 1. During the simulations, Arg118 initially recognizes the carboxyl oxygens of Neu5Ac, forms a strong hydrogen bond, and holds it. Then the galactose makes a tight interaction network through the oxygens O1, O2, O4, and O6 thus making a strong binding with lectin (Figure 8). WGA3 can also accommodate N23G in a different BM (BM2), which has lesser number of hydrogen bonds (4D, 3 W) when compared with BM1 (Table 2). The shift in BM from BM1 to BM2 is noted at 12 900 ps of the pair interaction energy plot (Figure 4(d)). BM1 has relatively 20.3 kcal/mol lower energy than BM2. The free energy calculations also propose that BM1 is 13.8 kcal/mol lower than BM2. From Table 2, it can be concluded that WGA3 shows better binding when compared with wild type and the other mutated lectins.

Figure 9. The distance between carboxylate oxygens of Neu5Ac and NH1/NH2 of Arg118 throughout the 20 ns molecular dynamics simulations in WGA3–N23G complex. This hydrogen bond persists up to 17.5 ns.

Twenty nanoseconds MD simulations are performed on the wild type WGA and mutated WGAs (WGA1, WGA2, and WGA3) to investigate the change in binding specificity of WGAs towards cell surface sialylglycans. In-depth analysis of trajectories and energy calculations propose that the double point mutated lectin WGA3 has the highest specificity towards the sialylgalactose with 15 hydrogen bonds of which eight are contributed by mutant residues Asn114 (S114N) and Arg118 (G118R). The key interaction is the strong hydrogen bonds between Neu5Ac and the mutant Arg118 (Figure 9). The additional contribution to the specificity is from the other mutant Asn114. WGA1 has higher specificity towards sialylgalactose than the wild type and the double point mutant WGA2. The key contribution to the best BM is from the mutant Asn114 (S114N), which contributes to five hydrogen bonds along with the hydrophobic cluster formed between Neu5Ac and Asn114, Cys117. WGA2 shows the slightly improved binding specificity towards N23G than wild type WGA. The mutant Lys118 (G118K) contributes three hydrogen bonds with N23G. The high mobility of the side chain of the mutant (Lys118) in WGA2 is accounted for the lesser specificity than the other mutated WGAs (WGA1 and WGA3). The wild type WGA shows three BMs, whereas the mutated WGAs have only two BMs. A summary of possible BMs and the corresponding relative energies, number of hydrogen bonds, hydrophobic interactions observed, and the binding free energy using MM-PBSA for the complexes WGA, WGA1, WGA2, and WGA3 with N23G are given in Table 2.

CONCLUSIONS

490

Figure 8. Atomic level interactions in BM1 of WGA3–N23G complex.


Wheat germ agglutinin exhibits significant binding specificity towards terminal sialylglycans occurring at the cell surface. The specific recognition and binding of WGA towards sialylglycans influence their role in induced apoptosis of tumor cells, blood group specificity, and cancer biomarkers. Because of the extensive importance of WGA binding to the sialylglycans, in the present study, the change in the binding specificity of mutated WGAs towards sialylgalactose is investigated using theoretical calculations. Wild type (WGA), single point mutated (WGA1), and double points mutated (WGA2 and WGA3) lectins


J. Mol. Recognit. 2014; 27: 482–492

BINDING SPECIFICITY OF MUTATED WGAS TOWARDS SIALYLGLYCANS in complex with N23G are simulated for 20 ns, and the change in binding specificity towards sialylgalactose has been examined using energy calculations. Double point mutations (S114N and G118R) increase the hydrogen bonding interactions between the receptor N23G and WGA3. The mutant Arg118 (G118R) in the double point mutation increases the specificity of WGA3 towards N23G because of its notable interaction with the carboxyl oxygen of Neu5Ac. The other double point mutation (S114N and G118K) in WGA2 is comparably weaker than the single point mutation. In general, the mutations of amino acids in the binding site increase the binding affinity of WGA lectins towards the sialylgalactose when compared to the wild type. The interaction patterns and the energy calculations (total pair wise interaction energy and the binding free energy using MM-PBSA) suggest that the binding affinity is of the order WGA3 WGA1 > WGA2 > WGA. Because the WGAs play a wide spectrum

of roles such as biomarkers in cancer discovery and therapeutics, and because of the roles of sialylglycans in the recognition phenomena, the present study would help in designing biomarkers experimentally with extended affinity and can be used in the development of glycan-based drugs for the treatment of diseases like cancer.

Acknowledgements PP and VM acknowledge DBT, India—AIST, Japan bilateral project for Junior Research Fellowship (BT/IC/JAPAN(BI)/02/2010). JFAS acknowledges CSIR for Senior Research Fellowship, and all the authors acknowledge the use of DBT funded Bioinformatics Infrastructure Facility (BIF) at the Department of Physics, Manonmaniam Sundaranar University (BT/BI/25/049/2012).

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J. Mol. Recognit. 2014; 27: 482–492

Precipitation and carbohydrate-binding specificity studies on wheat germ agglutinin.

Conformational analysis on the wild type and mutated forms of human ORF1p: a molecular dynamics study.

agmatine antiporter AdiC.

Endocytosis of wheat germ agglutinin binding sites from the cell surface into a tubular endosomal network.

Expression and secretion of wheat germ agglutinin by Saccharomyces cerevisiae.

Microcalorimetric study of wheat germ agglutinin binding to N-acetylglucosamine and its oligomers.

Insights into ligand binding to PreQ1 Riboswitch Aptamer from molecular dynamics simulations.

Insights into molecular interactions between CaM and its inhibitors from molecular dynamics simulations and experimental data.

Selective loss of wheat germ agglutinin binding to agglutinin-resistant mutants of Chinese hamster ovary cells.

Partial characterisation of a wheat germ agglutinin and an albumin.

Inhibition of fungal growth by wheat germ agglutinin.

Investigation of arc repressor DNA-binding specificity by comparative molecular dynamics simulations.

Wheat germ agglutinin in wheat seedling roots: induction by elicitors and fungi.

Insights into the role of Asp79(2.50) in β2 adrenergic receptor activation from molecular dynamics simulations.

Insights into the unfolding pathway and identification of thermally sensitive regions of phytase from Aspergillus niger by molecular dynamics simulations.

Insights into the mechanism of C5aR inhibition by PMX53 via implicit solvent molecular dynamics simulations and docking.

Wheat germ agglutinin accumulation in coleoptiles of different genotypes of wheat. Localization by monoclonal antibodies.

Wheat-germ agglutinin is synthesized as a glycosylated precursor.

Insights into the Tunnel Mechanism of Cholesteryl Ester Transfer Protein through All-atom Molecular Dynamics Simulations.

Influence of Concanavalin A, wheat germ agglutinin, and soybean agglutinin on the fusion of myoblasts in vitro.

Differences in the redistribution of concanavalin A and wheat germ agglutinin binding sites on mouse neuroblastoma cells.

Analyzing machupo virus-receptor binding by molecular dynamics simulations.

In silico simulations of STAT1 and STAT3 inhibitors predict SH2 domain cross-binding specificity.

Detailed molecular dynamics simulations of human transferrin provide insights into iron release dynamics at serum and endosomal pH.