Journal of Molecular Structure 978 (2010) 220–228

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Journal of Molecular Structure

journal homepage: www.elsevier .com/locate /molstruc

Specific intermolecular interactions of conserved water molecules with aminoacids in the Galectin-1 carbohydrate recognition domain

Santiago Di Lella a, Ariel A. Petruk b, Diego J. Alonso de Armiño b, Rosa M.S. Álvarez b,c,*

a Departamento de Química Inorgánica, Analítica y Química-Física (INQUIMAE-CONICET), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires,Ciudad Universitaria, Pabellón 2, C1428EHA, Ciudad de Buenos Aires, Argentinab Instituto Superior de Investigaciones Biológicas (CONICET), Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, Chacabuco 461,T4000ILI San Miguel de Tucumán, Tucumán, Argentinac Instituto de Química Física, Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, San Lorenzo 456, T4000CAN San Miguel de Tucumán,Tucumán, Argentina

a r t i c l e i n f o

Article history:Received 1 December 2009Received in revised form 3 March 2010Accepted 3 March 2010Available online 6 March 2010

Keywords:Galectin-1Quantum chemical calculationsBinding energiesHydrogen-bonding interactions

0022-2860/$ - see front matter � 2010 Elsevier B.V. Adoi:10.1016/j.molstruc.2010.03.008

* Corresponding author. Instituto de Química FísQuímica y Farmacia, Universidad Nacional de TT4000CAN San Miguel de Tucumán, Tucumán, Argent

E-mail address: mysuko@fbqf.unt.edu.ar (R.M.S. Á

a b s t r a c t

Water molecules, rigidly associated to protein surfaces, play a key role in stabilizing biomolecules andparticipating in their biological functions. Recent studies on the solvation properties of the carbohydraterecognition domain of Galectin-1 by means of molecular dynamic simulations have revealed the exis-tence of several water sites which were well correlated to both the bound water molecules observedin the crystal structure of the protein in the free state and to some of the hydroxyl groups of the carbo-hydrate ligand observed in the crystal structure of the complexed protein. In this work, we present astudy using quantum mechanical methods (B3LYP/6-311++G(3df,3dp)//B3LYP/6-31+G(d)) to determinethe energy involved in the binding of these water molecules to specific amino acids in the carbohydraterecognition domain of the protein. By modeling the hydroxyl groups of the carbohydrate by methanol,the energies associated to the local interactions between the ligand and the protein have been evaluatedby replacing specific water molecules with methanol. The values of the binding energies have been com-pared to those previously obtained by the molecular dynamic method.

� 2010 Elsevier B.V. All rights reserved.

1. Introduction

The presence of conserved water molecules in the structure ofbiomolecules has been subject of several experimental and compu-tational investigations for a long time. However, the role of thesewater molecules has not been well established yet and it is still acontroversial issue. What is clear is that the positions and orienta-tions that the water molecules adopt on the surface of proteinstructures play a key role in the stabilization and function of thebiomolecules. Sometimes, these water molecules, rigidly associ-ated to the binding sites should vacate their positions in order toallow proper binding, while others keep their coordinates as theybridge the interaction between the protein and the ligand [1–4].Bound water molecules in the proteins are often entropically orenergetically unfavorable because of the constrained orientationsand positions imposed by the protein surface, or due to a limitednumber of hydrogen bonds they are able to form when solvatingthe protein surface. Then, the displacement of water molecules

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lvarez).

from the active site by the ligand is one of the main sources ofbinding free energy. Even when the thermodynamic contributionof water to the binding process is still not completely understood,important and valuable progress has been achieved in the lastyears by using several techniques based on molecular dynamicsimulations. These studies have allowed a better understandingof the binding affinities of the ligands for a given protein fromthe evaluation of the properties of the solvent in the binding site[5–9]. Quantum mechanical methods, employing density func-tional theory with diffuse functions, have also been applied tothe study of the role played by conserved water molecules onthe protein surface [10]. Optimized geometries and structures ofhydrogen-bonded complexes in good agreement with previousMP2 calculations [11,12] and experimental data [10], as well asquantification of the binding energies resulting from specificwater–protein interactions were possible.

The structure and dynamic of water molecules in the carbohy-drate recognition sites of lectins has been subject to special studydue to the presence of hydrophilic regions that accommodate thehydroxyl groups of the ligand whether, by displacing water mole-cules or by forming complex hydrogen-bond networks with them.In this context, the solvation properties of the carbohydrate recog-nition domain (CRD) of human Galectin-1 (hGal-1), a galactosil–

S. Di Lella et al. / Journal of Molecular Structure 978 (2010) 220–228 221

terminal sugar binding soluble protein that plays a key role as amodulator of cellular differentiation and immunological response[14–16], have recently been investigated by MD simulations [17].The CRD, conformed by His44, Asn46, Arg48, His52, Asn61,Trp68, Glu71, and Arg73, presented eight well-defined regions,called water sites (ws), that showed a maximum water occupancyprobability in interaction with the protein. Ws, determined bycomputing several distribution functions for the water moleculesclose to specific hydrogen-bonding donor/acceptor atoms of theamino acid side-chains, were characterized in terms of water resi-dence times, interaction energies, and free energy contributions.These ws evidenced a close correspondence with the bound watermolecules observed in the crystallographic structure of hGal-1[18]. In addition, this correspondence was also observed for the li-gand-bound protein state, since the crystal structure of the com-plex showed that three of these well-ordered water moleculeswere displaced into the bulk while their positions were occupiedby the oxygen atoms of the carbohydrate ligand [17,18].

The focal point of the present study is to complement the pre-vious computational results based on MD simulations by predict-ing, at a quantum chemical level, the energies and structuresthat characterize the specific interactions between the water mol-ecules and the amino acids of the CRD of hGal-1. The most proba-ble locations and orientations adopted by the structural watermolecules were rationalized in terms of the number and strengthof the hydrogen-bond interactions that each water molecule is ableto form with the protein. This study was extended to the proteincomplexed with N-acetyllactosamine (LacNAc) by modeling thepresence of the OH groups of the sugar by methanol moleculesand the N-acetyl group by N-methylacetamide [10]. The resultswere associated with the impact that the water replacement bythe carbohydrate ligand exerts on the structure and energy of theCRD in the binding process.

2. Methodology

The Cartesian coordinates for the human Galectin-1 in both thefree state and complexed with N-acetyllactosamine were retrievedfrom the Protein Data Bank, codes 1W6N.pdb and 1W6P.pdb,respectively [18]. Hydrogen atoms were automatically added tothe X-ray structure by using the GaussView 3.09 Program. Accord-ing to the information obtained from the crystal structure, as wellas from the simulations of the ligand-free protein, eight conservedwater molecules strongly interact with His44, Asn46, Arg48, His52,Asn61, Trp68, Glu71 and Arg73, present in the CRD [17,18]. For ourpurposes, the protein structure was simplified to contain only theamino acids 28–74 conserving the original architecture of the CRD.The key amino acids involved in hydrogen-bonding interactionswith bound water molecules were then reduced to fragments con-taining only the side-chains. The first step to investigate thestrength of the interactions between each water molecule andthe protein consisted in geometry optimizations for all the possiblehydrogen-bonded dimer structures conformed by one water mole-cule and one amino acid fragment, extracted from the PDB files.Then, optimizations of trimers and larger clusters were performedaccording to the number of hydrogen-bonding interactions thateach water molecule was able to carry out with the neighboringamino acids. The B3LYP level of theory with a 6-31+G(d) basisset, employing the Gaussian 03 suite of program [19], was usedfor the optimizations. Subsequent frequency calculations were per-formed at the same level of theory to ensure all the structures wereminima and to determine the zero-point vibrational energies(ZPVEs). In all the cases, the most stable conformations were com-pared to the original crystallographic structures. When significantdistortions of the molecular systems resulted from the calcula-

tions, the main-chains of the fragments and short segments ofthe polypeptide chain were included. The optimizations of theselarger systems were performed using the Gaussian’s ONIOM meth-od [20] with the B3LYP/6-31+G(d): UFF model chemistries. Thus,the polypeptide chain, treated at a Molecular Mechanics levelmade it possible to maintain the configuration of the amino acidfragments around the conserved water as that adopted in the crys-tal structure. Those water molecules that appeared displaced bythe OH groups of the carbohydrate in the crystallographic structureof the complex were substituted by methanol molecules in the cor-responding dimers and clusters. In addition, two water moleculesthat appeared bridging the interactions between the ligand andthe protein in the complex crystal structure were evaluated byadding a methanol or N-methylacetamide molecule to the cluster.Optimizations and frequency calculations were performed at thesame level of theory used for the water-containing systems.

Additional single-point energy calculations were performed forall the optimized structures with the 6-311++G(3df, 3dp) basis set,including the counterpoise keyword in the route section. Bindingenergy of a water/methanol molecule to a single amino acid frag-ment or to a cluster of amino acid fragments was obtained withthe counterpoise correction [21], followed by the ZPVE and therelaxation corrections. The ZPVE values were scaled by using theempirical factor of 0.9775 [22]. Relaxation energies were calcu-lated to take into account the structural changes observed betweenthe isolated fragments and those in the clusters [23].

3. Results and discussion

Five of the eight structural water molecules observed in thecrystal structure of the ligand-free protein were considered of rel-evant importance in the binding process, since three of them weredisplaced by the carbohydrate while others two were involved inprotein–water–ligand interactions. The three remaining watermolecules did not evidence any change upon ligand binding.Fig. 1 shows the representative fragments of the interacting aminoacids delimiting the CRD of the protein in both the ligand-free stateand the complexed with N-acetyllactosamine, together with thestructural water molecules that were in good concordance withthe ws determined from the MD simulations. In the ligand-boundstructure the positions of the hydroxyl groups OH-4, OH-6 of gal-actose and OH-3 of glucose correspond to the three displacedwater molecules W1, W2 and W3. The presence of these conservedwater molecules was explained on the basis of the hydrogen-bondinteractions with several acceptor and donor groups of the aminoacids of the CRD [17]. However, some water molecules were alsointeracting with other side-chains not belonging to the CRD andbackbone atoms, as well as with other crystallographic water mol-ecules forming a complex hydrogen-bonding network [18].

3.1. Single hydrogen-bonded water/methanol–amino acid systems

Each of the single hydrogen-bonding interactions that a watermolecule is able to carry out with the surrounding amino acidside-chains was individually evaluated. Fig. 2 compares the place-ment of water molecules in the optimized water–amino acid di-mers, with the positions they had in the crystal structure of theprotein. In general, in order to allow a proper binding, water mol-ecules show significant displacements from their original relativelocations. For instance, in the W1–Arg48 and W8–Arg73 dimers,the minima corresponds to structures where the water moleculesare positioned equidistant to both the NH and NH2 groups of argi-nine, establishing simultaneous hydrogen-bonds with them(Fig. 2e). A similar situation was observed for the W4–Asp54

Fig. 1. (a) Representative fragments of the amino acids conforming the CRD of hGal-1, along the eight conserved water molecules in interaction with the amino acids nearby.(b) Representative fragments of the amino acids conforming the CRD of h-Gal-1 complexed with N-acetyllactosamine, along five conserved water molecules in interactionwith the amino acids nearby. In the ligand-bound state, W1–3 were replaced by the hydroxyl groups O4, O6 of galactose and O3 of N-acetylglucose fragment, while thehydroxyl group of C3 galactose participates in indirect hydrogen-bond with the protein through W50 and the N-acetyl group via W8-mediated interactions.

222 S. Di Lella et al. / Journal of Molecular Structure 978 (2010) 220–228

(Fig. 2l). These cyclic conformations agree with previous studies ofrelated systems [13,24].

On the contrary, small changes in the oxygen coordinates of thewater molecules are predicted for W1–His44, W2–Asn61, W3–Arg48, W2–Glu71 and W3–Glu71, (Fig. 2a, d, e, m, and n, respec-tively) suggesting that the crystallographic positions adopted bythe water molecules obey exclusively to the specific interactionswith the amino acids, individually. However, as it will be discussedlater, in all the cases water positions and orientations are the resultof the complex hydrogen-bonding networks determined by thepresence of the various donor and acceptor atoms belonging tothe close amino acid side-chains.

Table 1 lists the energies and the ZPVE corrections calculated forsingle water and methanol molecules, as well as for each of theinteracting amino acid fragments of the CRD. Table 2 lists the coun-terpoise corrected energy (EBSSE) of each optimized water–aminoacid dimer, along the energies of each monomer in the dimer,the corresponding relaxation energies, the ZPVE total corrections,and the final binding energies. The interaction energies calculatedfrom MD simulations of individual solvated amino acids [17] areincluded for comparisons. The RMSD calculated for the heavyatoms between the optimized dimer and the crystal structure is in-cluded in Table 2.

The binding energies calculated for the water-dimers are classi-fied in three groups: (i) the least favorable hydrogen-bondinginteractions correspond to dimers involving uncharged amino acidfragments, like histidine, asparagine, and tryptophan, with bindingenergies ranging between �3.07 and �5.82 kcal/mol; (ii) moreenergetically favorable than neutral amino acids are the associa-tions with positively charged amino acid side-chains. Thus, a bind-ing energy of �9.78 kcal/mol is calculated for a single interactionbetween water and arginine, while 12.33/12.51 kcal/mol is ob-tained when the water molecule acts as double donor yielding acyclic dimer. The more localized charge in lysine is responsiblefor the highly favorable interaction of �14.80 kcal/mol; (iii) thestrongest hydrogen-bonding interactions are obtained for watermolecules bound to the negatively charged glutamate and aspar-tate side-chains, with binding energies ranged from �13.44 to�15.36 kcal/mol.

Upon substitution of the water molecules W1–W3 by methanol,stronger interactions result since the hydrogen-bond lengths de-crease in 0.01–0.03 Å. No further differences in the dimer geome-tries are estimated. This modest geometrical difference isassociated with the favorable average increment of 1.0 kcal/molin the binding energy when methanol is the ligand, relative towater, in single hydrogen-bonding interactions, and 1.95 kcal/mol in double or single interactions with charged amino acids.The only exception of the unchanged geometries upon methanolsubstitution was the MeOH3–Glu71 dimer, where the oxygen atomof methanol experiences a significant shift towards one of the OEatoms of glutamate. The resulting single hydrogen-bond interactionis stronger and approximately 0.3 Å shorter that the correspondingto water interactions in W3–Glu71; however, the binding energyof methanol is only 0.96 kcal/mol more favorable than the corre-sponding to the binding of water, due to the decrease in the numberof hydrogen-bonding interactions. On the contrary, cyclic dimers areformed when methanol binds to arginine. As expected, in bothMeOH1–Arg48 and MeOH3–Arg48 dimers the calculated bindingenergies are 1.94–1.98 kcal/mol more favorable than the bindingof a water molecule to arginine 48. The corresponding binding ener-gies for methanol–amino acid dimers are included in Table 2.

3.2. Clusters of amino acids involving one water/methanol molecule

The most probable orientation and equilibrium position thateach water molecule adopts in the CRD are determined by consid-ering simultaneously all the possible hydrogen-bond interactionswith the closer donor and acceptor groups of the surrounding ami-no acids. Geometry optimizations and water binding energies arecalculated for clusters containing a conserved water moleculeand two or three amino acid fragments. Table 3 compares the dis-tances between the water oxygen and the heavy atoms involved ineach hydrogen-bond, previously obtained from the X-ray diffrac-tion and MD simulations of the ligand-free state [17,18] with thedistances resulting from the quantum chemical optimizations ofthe water–amino acid clusters. The binding energies of water tothe clusters are included and compared to the interaction energiesderived from the dynamic simulations.

Fig. 2. Optimized water–amino acid dimer structures, with the corresponding hydrogen-bond distances (Å) and angles (in degrees). The circles represent the originalpositions of the oxygen atoms of the water molecules, as they appeared in the crystal structure, and the water molecules represented by the stick-and-ball models correspondto the optimum coordinates to allow the proper water binding.

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According to both, the crystallographic structure and the MDsimulations, W1 is rigidly tied to the protein by interactions withthe NE2 group of histidine 44, the OD1 of asparagine 46, and theNH2 group of arginine 48 (Fig. 1). The optimized cluster showsthe side-chains of His44, Asn46 and Arg48 in positions comparableto those in the crystal, and the water molecule slightly shifts to beproperly hydrogen-bonded to the three amino acids simulta-neously Fig. 3. The three-coordinated W1 molecule is acting asdouble acceptor and single donor of hydrogen bonds. The compar-ison of the hydrogen-bond lengths to those calculated for the

corresponding dimers concludes that the interaction with His44results in a poor contribution to the binding of water to the cluster.In addition, the hydrogen-bond interaction between the NH groupof Arg48 and the carbonyl group of Asn46 in the optimized clustercontributes to dissipate the positive charge on arginine, with theconsequent negative effect on the total binding energy of waterto the cluster.

The observed position of W2 in the crystal structure and in thesimulations indicates that the water molecule is hydrogen-bondedto both the ND2 and OE1 groups of asparagine 61 and glutamate

Table 1Zero-point energy corrections and energies corresponding to the minima predictedfor water, methanol, N-methylacetamide, and each of the amino acid fragmentsparticipating in the hydrogen-bond interactions.

Molecule/amino acid fragment ZPVEa,b Energyb,c

Water 0.021094 �76.46438Methanol 0.051299 �115.77377Histidine 0.098853 �265.63431Asparagine 0.073542 �209.30329Arginine 0.116776 �245.17631Glutamate 0.076796 �267.94500Aspartate 0.048144 �228.61713Lysine 0.108302 �135.59184Tryptophan 0.157464 �403.27294N-methylacetamide 0.102126 �248.62386

a B3LYP/6-31+G(d); scaled values by 0.9775.b In hartrees.c B3LYP/6-311++G(3df, 3dp).

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71, respectively (Fig. 1). As can be seen in Fig. 4, the optimized rel-ative positions of most of the non-hydrogen atoms belonging tothese amino acids and the water oxygen remain without signifi-cant changes in comparison to those in the crystal. The orientationadopted by the water molecule in the W2–Asn61-Glu71 trimer isconsistent with effective interactions with both amino acid side-chains, while the shortening of the hydrogen-bond lengths is inagreement with the magnitude of the resulting binding energy(Table 3).

In the crystal, W3 appears strongly interacting with both NHatoms of Arg48 and with both OE atoms of Glu71. When the inter-actions of the water molecule are analyzed with these amino acids

Table 2Energies of the water–amino acid and methanol–amino acid dimers (EBSSE), monomer (Emo

(ZPVEt), relaxation energies (Erelax), the corresponding corrected binding energies (BE), anatoms between the optimized and the crystal structures are included.

Dimer EBSSEa Emonomer

a Efragmenta

W1–His44 �342.10603 �76.46434 �265.63415W1–Asn46 �285.77790 �76.46404 �209.30321W1–Arg48 �321.66382 �76.46427 �245.17595

W2–Asn61 �285.77391 �76.46434 �209.30306W2–Glu71 �344.43450 �76.46267 �267.94464

W3–Arg48 �321.66413 �76.46427 �245.17588W3–Glu71 �344.43773 �76.46217 �267.94482

W4–Asn46 �285.78113 �76.46381 �209.30261W4–His52 �342.10601 �76.46434 �265.63416W4–Ser29 �192.24574 �76.46414 �115.77368W4–Arg48 �321.65927 �76.46431 �245.17557

W5–His52 �342.10602 �76.46434 �265.63414

W6–His44 �342.10911 �76.46398 �265.63431W6–Asn33 �285.78113 �76.46380 �209.30261

W7–Trp68 �479.74422 �76.46435 �403.27272W7–Lys63 �212.08232 �76.46426 �135.59131

W8–Arg73 �321.66382 �76.46427 �245.17595W8–His52e �285.78113 �76.46381 �209.30261W8–Asp54 �305.10999 �76.462142 �228.61679

MeOH1–His44 �381.41685 �115.77368 �265.63410MeOH1–Asn46 �325.08745 �115.77346 �209.30321MeOH1–Arg48 �360.97534 �115.77325 �245.17570MeOH2–Asn61 �325.08480 �115.77370 �209.30324MeOH2–Glu71 �383.74550 �115.77158 �267.94458MeOH3–�Arg48 �360.97563 �115.77324 �245.17556MeOH3–Glu71 �383.74656 �115.77094 �267.94482

a In hartrees.b In kcal/mol.c Ref. [17].d In Å.e Hydrogen-bond interaction with the main-chain carbonyl of His52.

individually, good correlations are obtained among the positionsadopted by the water oxygen in the experimental and simulatedprotein structures (Fig. 2g and n), as well as a fairly good concor-dance between the estimated binding energies and the interactionenergies derived from the MD simulations (Table 2). However, sev-eral attempts to obtain the minimum structure of the W3–Arg48-Glu71 trimer containing the water molecule located in a compara-ble localization to that in the crystal failed, indicating that thecoordinates of all the involved non-hydrogen atoms observed inthe crystal structure of the ligand-free protein, as well as the posi-tion of the corresponding ws derived from the dynamic simula-tions are likely to be determined by the global architecture of theCRD, that are computationally expensive at the level of theory usedin this study. The interaction of the water molecule with both ami-no acid side-chains is optimized to a cyclic trimer formed by threehydrogen-bonds. The bi-coordinated water molecule participatesbridging the NH1 and OE1 atoms through strong hydrogen-bonds,yielding a calculated binding energy of �17.25 kcal/mol for water,(Fig. 5). A more favorable binding energy would be expected takinginto account the interactions of the water molecule with twocharged amino acids. However, the hydrogen-bond interaction be-tween arginine and glutamate exerts a negative effect on the waterbinding energy. The difference with the reported interaction en-ergy (DE = �20.09 kcal/mol) [17] could be associated to the distor-tion predicted for this cluster in relation to the position of the ws inthe simulated protein structure.

The water molecules W1, W2 and W3 are replaced by methanolin the respective arrangements of amino acid fragments, and thecorresponding optimized structures and binding energies are cal-culated. In general, good correlations are obtained between the

nomer) and fragment (Efragment) energies in dimers, zero-point total correction energiesd the interaction energies (DE) obtained from MD simulations. RMSD for the heavy

ZPVEtb Erelax

b BEb DEc RMSDd

1.21 0.13 �3.39 �6.34 0.222.05 0.27 �4.37 �4.03 0.562.18 0.30 �12.34 �12.01 0.65

1.47 0.17 �2.44 – 0.072.02 1.30 �13.74 �13.92 0.26

2.20 0.34 �12.51 �12.01 0.132.59 1.50 �15.20 – 0.29

2.62 0.79 �5.82 �6.23 0.261.15 0.13 �3.44 �6.34 0.702.12 0.21 �2.64 – 0.351.89 0.50 �9.78 �10.38 1.15

1.27 0.14 �3.32 �6.34 0.27

1.92 0.25 �4.61 �7.52 0.712.62 0.79 �8.02 �6.23 0.45

1.27 0.15 �3.07 �3.82 0.381.58 0.41 �14.80 – 0.42

2.18 0.30 �12.33 �12.01 0.902.62 0.79 �5.82 – 0.822.52 1.61 �15.36 – 0.72

0.97 0.19 �4.531.39 0.25 �5.131.57 0.71 �14.281.27 0.07 �3.581.12 1.64 �15.661.55 0.80 �14.491.28 1.89 �16.16

Table 3Distances obtained by quantum–mechanical optimizations (rQM) between the heavy atoms associated by hydrogen-bonding interactions in water–amino acid clusters, and thecorresponding water binding energies (BE). Comparison with the distances (rX-ray, rMD) and interaction energies (DE) obtained from MD simulations of the protein. Distancesobtained from the X-ray structure and RMSD for the heavy atoms between the optimized and the crystal structures are included. Distances and RMSD expressed in Å and energiesin kcal/mol.

Water Amino acid fragment Donor/acceptor atom rX-ray rMDa rQM RMSD BE DEa

W1 His44 NE2 2.69 3.04 3.12 0.47 �6.16 �6.62Asn46 OD1 3.24 2.89 2.75Arg48 NH2 3.11 3.02 2.86

W2 Asn61 ND2 2.88 3.00 2.85 0.35 �16.83 �15.19Glu71 OE1 2.74 2.75 2.59

W3 Arg48 NH1 2.87 2.96 2.70 0.81 �17.25 �20.09Arg48 NH2 3.25 3.01 3.74Glu71 OE1 2.86 2.93 3.60Glu71 OE2 2.76 2.73 2.59

W4 His52 NE2 3.46 3.09 2.88 1.06 �5.86 �3.54Asn46 ND2 3.56 3.10 3.81Asn46 OD1 3.24 – 2.97Ser29 OG 3.11 – 2.89

W6 His44 ND1 3.53 3.04 2.83 0.83 �8.75 �5.93Asn33 ND2 3.05 – 2.92

W7 Trp68 NE1 3.72 3.07 3.04 0.81 �10.64 �7.02Lys63 NZ 3.45 – 2.78

W8 Arg73 NH2 3.51 3.01 2.93 1.67 �4.78 �22.20His52b O 2.51 – 2.76

a Ref. [17].b Hydrogen-bond interaction with the main-chain carbonyl of His52.

Fig. 3. Optimized geometry for the cluster formed with W1–His44-Asn46-Arg48.The water molecule is hydrogen-bound to the three amino acids by singleinteractions with each of them. For comparison, the positions adopted by theheavy atoms of the truncated amino acids and the water in the crystal structure aresuperimposed. Hydrogen-bond distances (Å) between water–amino acid andmethanol–amino acid (in parentheses) are included.

Fig. 4. Optimized geometry for the cluster formed with W2–Asn61-Glu71. Thewater molecule is hydrogen-bound to both amino acids by single interactions witheach of them. For comparison, the positions adopted by the heavy atoms of thetruncated amino acids and the water in the crystal structure are superimposed.Hydrogen-bond distances (Å) between water–amino acid and methanol–aminoacid (in parentheses) are included.

S. Di Lella et al. / Journal of Molecular Structure 978 (2010) 220–228 225

optimized geometries and the crystallographic structure of theligand-bound protein. On the other hand, smaller shortening inthe hydrogen-bond lengths than those observed in the methanol-dimers, are estimated for the interactions in the MeOH2 andMeOH3 containing clusters. The hydrogen-bonding interactionsof methanol with histidine and arginine in MeOH1–His44-Asn46-Arg48 are stronger than the corresponding interactions of thewater molecule, since a shortening of 0.03 Å is estimated at thetime that the hydrogen-bond length between methanol and aspar-agine was larger by 0.04 Å. This behavior agrees with previous MDsimulations of Galectin-1–oligosaccharide complexes indicatesthat the position occupied by the oxygen of the water or the 4-hy-droxyl group of galactosil residue provides the binding specificityfor galactose due to the interactions with His44 and Arg48 [25].

In addition, since the OH-6 strongly interacts with Asn61 andGlu71, the intramolecular hydrogen-bond interaction with the ax-ial OH-4 is not possible; then, the interaction of OH-4 with the pro-tein is favored [26]. The energies of the methanol binding to theclusters His44-Asn46-Arg48, Asn61-Glu71, and Arg48-Glu71 are�8.21, �17.71 and �19.49 kcal/mol, respectively.

According to the MD simulations, the structural water moleculeW5 is one of the least tightly bound to the CRD in the ligand-freestate, forming a single hydrogen-bond to histidine 52 and evidenc-ing a high exposition to the bulk solvent. It is not possible to get theoptimized localization and orientation of this water molecule inthe CRD by quantum chemical calculations. Then, in this case,the binding energy of this molecule to the free protein could notbe determined. The crystal structure of the complexed protein

Fig. 5. Optimized geometry for the cluster formed with W3–Arg48-Glu71. Thewater molecule is hydrogen-bound to both amino acids by single interactions witheach of them. For comparison, the positions adopted by the heavy atoms of thetruncated amino acids and the water in the crystal structure are superimposed.Hydrogen-bond distances (Å) between water–amino acid and methanol–aminoacid (in parentheses) are included.

Fig. 6. Schematic representation of the optimized geometry for the cluster formedwith W50 , W4, Arg48, Asn46 and a methanol molecule modeling the OH-4 ofgalactose.

226 S. Di Lella et al. / Journal of Molecular Structure 978 (2010) 220–228

shows a water molecule analogous to W5 in the free protein. Thiswater (W50) shows strong interactions with the ligand–proteincomplex via an extra hydrogen-bond with OH-3 of galactose. Acluster containing the representative fragments of asparagine 46and arginine 48, with the conserved W50, W4, and a methanol mol-ecule, that substituted the hydroxyl group of the ligand, is opti-mized. A schematic representation of the resulting geometry,together with the complex hydrogen-bond network that fixes thecoordinates of W50 is shown in Fig. 6. The energies calculated forthe binding of the ligand, W4 and W50 to this cluster are �8.55,�4.22, and �1.72 kcal/mol, respectively. Strong interactions ofW50 with other water molecules of the solvent would be theresponsible for the low negative binding energy, according to Liand Lazaridis [6].

The crystal structure and the MD simulations of the free proteinagreed with the presence of a conserved water molecule (W8)hydrogen-bonded to arginine 73. However, discrepancies arose be-tween the experimental and the simulated complex structures.Thus, in the crystal structure, W8 was conserved by mediating

interactions between the protein and the NH group of the N-acet-ylglucose fragment, while the MD simulations predicted thereplacing of W8 by the carbonyl oxygen of the N-acetyl group. Asimilar situation had been addressed for Concavalin A complexedwith a synthetic analog of the natural ligand in which a hydroxy-ethyl side-chain was added [8]. In the reported case, earlier MDsimulations had pointed out that, upon ligand binding specificbound water was displaced [3], while the crystal structure indi-cated that, although the ligand perturbed the position of the con-served water, it did not displace it [8]. By simulating thepresence of the ligand with N-methylacetamide, a comparableoptimized geometry to that observed in the crystal structure ofthe complexed hGal-1 is obtained for the cluster involving W8,with a RMSD of 0.89 Å. Fig. 7 shows the optimized cluster contain-ing the conserved water molecule in both states of the protein. Thecalculated binding energies of W8 to the ligand-free and ligand-bound protein are �4.78 and �15.42 kcal/mol, respectively. Thelarge difference between theses values obeys to two main factors:(i) the presence of the ligand increases the number of hydrogen-bonds with W8, and (ii) W8 is hydrogen-bonded to aspartate 54in the complex; the interaction with this negatively charged aminoacid is the major contributor to the total binding energy of W8 tothe cluster. On the other hand, a substantial difference is also ob-served between the interaction energy calculated from simulationsand the binding energy of W8 to the ligand-free protein (Table 3).In addition to the large geometrical distortion predicted in refer-ence to the original structure, this fact is rationalized on the basisthat this water is located on the external surface of the CRD, andshows a high probability of interaction with other water moleculesof the solvent. Their effect is not taken into account in the totalbinding energy calculation. Geometry optimizations performedon the structure containing the replacing carbonyl oxygen of theN-acetyl group in the position of W8, as it was predicted by MDsimulations, are not successful since large distortions of the clusterare estimated.

The positions of W4, W6, and W7 in the CRD do not show sig-nificant changes upon ligand binding, as is observed in the crystal-lographic structures. For this reason they are considered non-relevant structural water molecules. In addition, the previous MDsimulations indicated that ws6 and ws7, and ws5 as well, werethe sites showing the lowest probabilities of finding a water mole-cule [17], according to the single hydrogen-bond interaction that

Fig. 7. Schematic representation of (a) the optimized structure of the cluster formed with W8, Arg73 and a fragment containing the peptidic bond between His52 (carbonylpart) and Gly53, in the free protein; (b) the optimized structure of the cluster formed with the same fragments and molecules from point (a), plus Asp54. The difference in thehydrogen-bonding network can be clearly seen.

S. Di Lella et al. / Journal of Molecular Structure 978 (2010) 220–228 227

each of them form with the amino acids of the CRD (Fig. 1) [6]. Inorder to minimize the large geometrical distortions observed in theoptimized W6–His44 and W7–Trp68 dimers (Fig. 2), these struc-tural water molecules are analyzed considering an additionalhydrogen-bond with a close external-CRD amino acid side-chain.Thus, optimizations and water binding energies are calculated forW6–His44-Asn33 and W7–Trp68-Lys63 trimers, and acceptableapproximations to the crystal water coordinates are obtained. Sim-ilarly, the hydrogen-bond interaction between W4 and the side-chain of serine 29 is included in the cluster W4–Asn46-His52. Asis expected, the corresponding energies for the water binding isoverestimated in relation to the interaction energies derived fromde simulations.

As it has been pointed out above, in the ligand-bound protein,the OH-3 group promotes the formation of a more complex hydro-gen-bond network that stabilizes the presence of W50 in the CRD,yet it reduces the number of interactions involving W4, leadingto a less negative binding energy of this water molecule in thecomplex.

4. Conclusions

Water molecules in the CRD of Gal-1 are able to act as eitheracceptor and/or donor atoms in hydrogen-bond formations; how-ever, from the magnitude of the interaction energies obtained, itis clear that water prefers to be a donor whether in single associa-tion with uncharged amino acid side-chains, by 0.98–1.93 kcal/mol, or forming cyclic dimers with charged amino acid side-chains,by the average 2.86 kcal/mol. A similar trend is observed for theinteraction of methanol.

A thermodynamic study on protein–water–ligand interactionsindicates that the probability of finding water in a ws as well asthe solute–solvent interaction energy, correlate with the numberof hydrogen-bond formed between the water and the protein [6].Accordingly, the binding energies of W4 and W8 become lessand more favorable, respectively, in the complexed protein com-pared to the ligand-free state. However, the results obtained forthe replaceable water molecules (W1, W2 and W3) do not fullyagree with these assessments, since more favorable binding ener-gies are obtained for W2 and W3. These appear forming twohydrogen-bonds with the proteins while W1 is hydrogen-bondedto three acceptor/donor groups.

This study clearly demonstrates that the presence of highly con-served water molecules in the protein structure obeys not only tothe number of hydrogen-bonds that they establish with the proteinbut also to the strength of these interactions. The strength of thewater–amino acid interactions depends, in turn, on the specificcapabilities as acceptor/donor of hydrogen atom of the amino acids

in the water neighborhood, the equilibrium position of the watermolecule with respect to these amino acids, and the possible inter-actions among the participant amino acids. The best example ofthese joint effects acting on a given water molecule is the clustercontaining W1, where two consequences derives from the pres-ence of Asn46 in the interacting cluster: (i) it is key in maintainingthe position of the water molecule as is observed in the crystal andsimulated structures; otherwise, water migration towards an equi-distant position between two NH groups of arginine overcomingthe optimum interaction with histidine is observed; (ii) the totalbinding energy of W1 to the cluster is affected by the hydrogen-bonding interaction between Asn46 and Arg48.

A recent study that states the probability of finding water mol-ecules inside the ws is directly correlated to the likeliness of find-ing a hydroxyl group of the ligand in the complexed protein [5].Similarly, our findings show that the best correlations betweenthe crystal and the optimized structures are obtained for the clus-ters including W1 and W2, which indeed occupy critical positionsfor ligand binding [17,18]. Furthermore, they correspond to wswith the highest probabilities, and their binding energies showgood concordances with the reported interaction energies [17].

The most favorable binding energies obtained upon replacingwater molecules with methanol are consistent with the substitu-tion of the conserved water molecules by the hydroxyl groups ofthe galactosyl residue during the binding process.

Acknowledgments

This work was supported by Grants from Consejo Nacional deInvestigaciones Científicas y Técnicas (CONICET) and Consejo deInvestigaciones de la Universidad Nacional de Tucumán (CIUNT).R.M.S.A. is member of the Research Career of CONICET. S.D.L. is aPost Doctoral Fellow of CONICET. A.A.P. is a Doctoral Fellowshipof CONICET.

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