MOLECULAR DOCKING OF NANOSIZED TITANIUM DIOXIDE MATERIAL TO THE EXTRACELLULAR PART OF GABAB-RECEPTOR

A spatial model of nanosized titanium dioxide material was created using Discovery Studio Visualizer software, versions 2.0 and 2.5. A search for and analysis of possible sites of its docking to the extracellular part of GABAB1а receptor subunit were performed using the algorythm for molecular docking PatchDock. The dimensions of the obtained ТіО2 nanoparticle surface were (18.925 × 3.785 × 19.028) Å. Four potentially possible sites of ТіО2 docking to the extracellular part of GABAB1а receptor subunit of GABAB were identified. The ТіО2 nanoparticle demonstrated high affinity of docking to one of the receptor sites with the geometric shape complementarity score of 12562, taking the following values in other sites: 10746; 10370; 10204. The approximate interface area of complex of the extracellular part of GABAB1а receptor subunit of GABAB with ТіО2 for the site with the highest geometric shape complementarity score was 1949.80 Å, and for others – 1273.20 Å, 1261.10 Å and 1170.30 Å, respectively. The evaluation of аtomic contact energy demonstrated the following values for the sites of ТіО2 nanoparticle docking: 362.92; 173.93; 340.63 and 224.61. The nature of connections, stabilizing the sites of ТіО2 docking to the extracellular part of GABAB1а receptor subunit of GABAB, was analyzed in accordance to their amino acid composition.


INTRODUCTION
It is known that in the form of nanoparticles, many substances acquire fundamentally new physical and chemical properties and are quite active in that state regarding the biological objects, whose macromolecules are also at the level of the nanometer dimensions, revealing their function via the formation of supermolecular complexes. These materials include titanium dioxide (ТіО 2 ), whose use in the form of nanoparticles has considerably extended in modern highly technological production processes in different sectors of economy, in particular, in food production and pharmacology [21]. According to literature data [3,4,5,14,27], ТіО 2 is a polar adsorbent, whose most active centers are superficial hydroxyl groups, capable of dissociating and forming positively and (or) negatively charged adsorption centers. It was shown [18] that ТіО 2 particles are capable of adsorbing on its surface aminoacids, in particular, histidine (Fig. 1).

Fig. 1.
Histidine adsorption on the surface of ТіО 2 nanoparticles [18]. Amino acids are bound to the surface of ТіО 2 , which creates conditions for intramolecular transfer of a proton onto the carboxyl group of adsorbed histidine. In addition, the imidazole ring may interact via π-orbitals with titanium atoms, located on the surface, and the amino acid may form H-bonds with superficial atoms of oxygen [4] Рис. 1. Адсорбція гістидину на поверхні наночастинок діоксиду титану [18]. Аміногрупи зв'язуються з поверхнею ТіО 2 , що створює умови для внутрішньомолекулярного перенесення протона на карбоксильну групу адсорбованого гістидину. Крім того, імідазольне кільце може взаємодіяти з атомами титану, що містяться на поверхні, через π-орбіталі, а аміногрупа може формувати H-зв'язки з поверхневими атомами оксигену [4] Taking that into consideration, it was interesting to investigate molecular mechanisms of interaction between ТіО 2 nanoparticles and molecules of proteins-receptors, in particular, metabotropic GABA B receptor [2,11] whose the mediator is a known transmitter of the central nervous system, γ-aminobutyric acid, that participates in control over blood circulation, implemented by neurons of dorsomedial and ventrolateral divisions of medulla oblongata [23,24,26]. To solve this issue by the molecular docking method, the search for binding sites of ТіО 2 particles and the extracellular part of GABA B receptor was performed. The aim of our work was also to construct the spatial structure of ТіО 2 particle, using Discovery Studio Visualizer software 2.0 and 2.5 of versions. The construction of the spatial structure of full-size GABA B receptor (Fig. 2), the investigation of its molecular dynamics and estimation of the energy of nonvalent interactions in the model of the complex of GABA B receptor with lipid bilayer membrane was performed in our previous work [19].

MATERІALS AND METHОDS
In order to construct a spatial structure of ТіО 2 nanoparticle, its crystal modification, anatase, was used [10]. A remarkable specificity of the modification of this nanoparticle material is a system of channels, oriented parallel to the plane of the crystallographic face. The channels in the cross section form a square with the side of 3.35 Ǻ. The anatase structure has a form of three-dimensional chains made of ТіО 6 octahedra. Each octahedron contains a central ion Ті +4 surrounded by six anions О -2 . Four anions are located in the equatorial plane, and two -in the axial points. The equatorial connections

RESULTS AND DISCUSSION
According to the catalog, the receptor subunit of gamma-aminobutyric acid, type B, was indicated in the work as GABA B1a [1], where а relates to the subunit isoform, and 1 indicates its number. The spatial structure of the extracellular part of GABA B1a subunit (15-592 amino acid residues) was obtained via the molecular simulation of the full-size GABA B receptor, that was conducted in our previous studies (Fig. 3, А) [19]. The threedimensional structure of the ТіО 2 particle (Fig. 3, B) was simulated using Discovery Studio Visualizer software 2.0 and 2.5 of versions. Taking into consideration the data about the crystallography of anatase, the following parameters of the elementary cluster of ТіО 2 were used in our work: A = B = 3.785 Å; C = 9.514 Å, where А, В, С -lengths of the crystalline grid. a = b = γ = 90°, where a, b, γ -dimensions of crystal angles. The spatial symmetry group (the combination of symmetry transformations, remarkable for atomic structure of crystals) of titanium dioxide, is I4(1)/amd [7,12]. The dimensions of the surface of ТіО 2 nanoparticle obtained after the simulation of the spatial structure, were as follows: (18.925 × 3.785 × 19.028) Å.

Fig. 2.
Spatial structure of GABA B receptor. This is a heterodimer consisting of two subunits (GABA B1а and GABA B2 ) [22]. In its turn, each subunit consists of the extracellular part (β-elements successively interchanging α-helices forming unique "claws"); the transmembrane part (containing seven successively connected α-helices); the intracellular part (containing the part, that serves for the formation of the superhelical structure) [8,9,22] Рис. 2. Просторова структура ГАМК Б -рецептора. Це гетеродимер, що складається з двох субодиниць (ГАМК Б1а та ГАМК Б2 ) [22]. У свою чергу, кожна субоди ниця складається з зовнішньоклітинної частини (β-елементи, які послідовно чергуються з α-спіралями, утворюючи своєрідні "клешні"); трансмембранної частини (містить сім послідовно з'єднаних α-спіралей); внутрішньоклітинної частини (містить ділянку, що слугує для утворення надспіралізованої структури) [8,9,22]  A search for and analysis of the interaction sites of TiO 2 and the extracellular part of the GABAB 1а of GABA B receptor subunit was performed using PatchDock web-service. The input parameters for the docking were the PDB coordinate file for the extracellular part of the GABA B1a receptor subunit and the TiO 2 nanoparticle. Clustering of RMSD (Root-mean-square deviation) was chosen as 4.0 Å. Clustering of RMSD is a positive number that specifies the radius of the RMSD clustering in angstroms. This value is used in the final clustering stage of the algorithm. It ensures that the distance between any of two output solutions will be at least the specified clustering of RMSD value. The default value for this parameter is 4 Å [25]. Complex Type was chosen as protein-small ligand. In the case of protein-small ligand docking, the algorithm uses a parameter set optimized for small-size molecules. Potential binding sites of the receptor and the ligand, respectively, were not chosen. A web page that presents the solutions is automatically generated. The output of PatchDock is a list of candidate complexes between extracellular part of GABA B1a receptor subunit and TiO 2 . The geometric score (Score), the аtomic сontact energy (ACE) [28], the size of interface area (Area) and the actual rigid transformation of the solution for the four binding sites are shown in Table 1. The atomic contact energy is used to estimate the contribution of the desolvation energy to the scoring function. The ACE score of a pair of protein atoms is defined as the free energy difference between two protein-atoms and water contacts and the sum of a protein-atom to protein-atom contact and a water to water contact. Presented transformations are 3D transformations that include 3 rotational angles and 3 translational parameters. These transformations are applied on the TiO 2 nanoparticle.
The results of the molecular docking of the TiO 2 nanoparticle demonstrate that the latter induces four potential sites of different affinity for its binding to the extracellular part of GABA B1а of GABA B receptor subunit. According to data of the estimations of PatchDock web-service, TiO 2 possessed high affinity of binding to one of receptor sites with the geometric shape complementarity score of 12562, and in other sites -10746; 10370; 10204. The approximate interface area of the complex of the extracellular part of subunit GABA B1а of GABA B receptor and TiO 2 for one of sites is 1949.80 Å, and for others -1273.20 Å, 1261.10 Å and 1170.30 Å, respectively. This value is in distinct correlation with the geometric shape complementarity score. It was determined that the level of affinity as a result of strong intramolecular interaction in the site of the proteinreceptor binding TiO 2 , the distribution within the site of amino acids forming long-term electrostatic hydrogen bonds, hydrophobic interactions are determined by their location on the receptor molecule, namely, the first one -on the side, opposite the slit between the lobes 1 (LB1) and 2 (LB2) closer to N-terminal part, the second one -in the slit between LB1 and LB2, the third one -on the surface of the receptor macromolecule near the slit between LB1 and LB2 and the fourth one -on the side, opposite the slit between LB1 and LB2 closer to the zone of their binding. The sites were numbered according to the reduction in their affinity degree. It was established (Fig. 4) that the amino acid composition and environment of the first binding site include the following amino acids: Trp37, Glu38, Val74, Thr92, Arg95, Cys96, Arg98, Gly116, Gly117, Asp118, Leu119, Pro120, Leu122, Pro153, Lys154, Pro155, Hіs156, Arg258, Arg277, Gln278, Pro281, Gln503, Thr504, Asp507, Gln508, Tyr510, Arg511, Asn514, Asp528, Ala529, Ser530. The evaluation of the аtomic contact energy demonstrated its value for this site of ТіО 2 nanoparticle binding to be 362.92.
The analysis of the amino acid composition of the site with the highest affinity of binding ТіО 2 (the first site) demonstrated that the variety of its composition includes all currently known amino acids with polar charged radicals capable of forming long-term electrostatic charges (the charge of the amino acid radical is indicated in brackets): Glu38 (-), Arg95 (+), Arg98 (+), Asp118 (-), Lys154 (+), Hіs156 (+), Arg258 (+), Arg277 (+), Asp507 (-), Arg511 (+), Asp528 (-) with prevailing amino acids, whose radicals have a positive charge. Among these amino acids, arginine and lysine are amino acids with high occurrence degree and the least conformational entropy of the side chain (as the estimation of the amino acid ability of forming the helix), for which Gibbs free energy (∆G) is (-0.68) kJ/mol and (-0.65) kJ/mol, respectively (calculated via changes in the temperature of the helix-coil transition while injecting a specific amino acid into the standard amino acid sequence of peptide), according to literature data [20]. As for glutamic and asparagine acids which are in this site composition, at the expense of the carboxylic group of side chains they are well-adsorbed on the surface of anatase [15]. The stabilization of the position of ТіО 2 particle in this site of binding to the protein-receptor GABA B , in addition to the electrostatic interactions, is affected by the hydrogen bonds, whose formation, as seen from the model, is facilitated by the following polar uncharged amino acids: Thr92, Cys96, Gln278, Gln503, Thr504, Gln508, Tyr510, Asn514 and Ser530. ∆G of these amino acids is in the range of (-0.07) -(-0.35) kJ/mol. Noteworthy it is the presence of cysteine in the list of the mentioned amino acids, as high polarization capacity of the electronic envelope of its sulfur atom makes it an active center of interacting with reagents and, as may be assumed, -with ТіО 2 in particular. The amino acid composition of this binding site also contains the hydrophobic amino acids, including four residues of proline. It is known [13,20] that proline (∆G = +3 kJ/mol) is the only cyclic amino acid whose amino group is not free and is a part of heterocycle, so this amino acid does not participate in the formation of the hydrogen bond. Proline binds to the polypeptide chain via covalent bonds and forms its clear link with the open access of peptide bonds. According to literature data [18] regarding the capability of ТіО 2 to form bonds both with amino groups of amino acid residues in protein and to its peptide bonds, they may become an additional place of interacting with nanoparticles. As stated above, the affinity of the first binding site acquires the value exceeding the value of this parameter for all three following sites. The results of estimating the approximate interface area of complex for each binding site of TiO 2 allowed revealing similar regularities (Table 1). The following binding site (Fig. 5) for TiO 2 particle and its environment includes such amino acids: Gly181, Trp182, Cys246, Ser247, Ser248, Ser270, Ser271, Gln314, Thr316, Val318, Ser345, Tyr367, Glu368, Thr369, Arg372, Lys373, Trp395, Ala397, Ile403, Tyr404, Asp405, Pro406, Ser407, Glu459, Gly462, Gly463, Phe464, Gln465, Glu466. The estimations of ACE for this site performed using PatchDock web-server, demonstrated that it acquires the value of 173.93. Fixing of the TiO 2 nanoparticle in the second binding site located in the slit between LB1 and LB2 of the extracellular part of the receptor, also occurs with the participation of the electrostatic interactions, ensured by polar radicals of amino acids, whose number is almost twice smaller than in the first site located opposite the slit between LB1 and LB2 close to N-terminal part (Glu368 (-), Arg372 (+), Lys373 (+), Asp405 (-), Glu459 (-), Glu466 (-)), which may be related to a low, compared to aqueous environment, value of the dielectric constant of the environment in the "claws" of receptor macromolecule. This site has fewer amino acids with a small value of conformational entropy: only one amino acid, arginine, and one amino acid, lysine, whose Gibbs free ener gy, as stated above, is (-0.68 and 0.65 kJ/mol). ∆G for other polar charged amino acids of this binding site takes the values from (-0.15) to (-0.27) kJ/mol. The analysis of obtained results demonstrated that a considerable number of bonds with ТіО 2 , formed at the second binding site, are hydrogen bonds, whose formation is promoted by the radicals of polar uncharged amino acids (Cys246, Ser247, Ser248, Ser270, Ser271, Gln314, Thr316, Ser345, Tyr367, Thr369, Tyr404, Ser407, Gln465), among which the prevalence in amount is attributed to the capping amino acid serine which is known to be the most reactive amino acid due to its functionally active hydroxyl group [17]. As stated above, cysteine is another amino acid among polar uncharged ones whose reactive capability is ensured by the sulfur atom with high polarization capacity of its electronic envelope. ∆G for the abovementioned amino acids is in the range of (-0.17) to (-0.35) kJ/mol. On contrary to the first binding site of ТіО 2 , a group of the hydrophobic amino acids in its composition contains one amino acid proline instead of four. In terms of quantity, the number of the hydrophobic and non-polar amino acids in both sites is practically the same.
The comparison of the binding site with the lowest affinity (the fourth binding site), located opposite the slit between LB1 and LB2 closer to the zone of their connection. The binding site with the highest affinity demonstrates that on contrary to a second one, the content of polar uncharged amino acids, whose radicals participate in the formation of hydrogen bonds, is presented by two amino acids only: tyrosine and asparagine with the highest conformational entropy of the side chain (∆G takes the values of (-0.17 and -0.11) kJ/mol). As for polar uncharged amino acids, whose radicals form electrostatic interactions, when compared against the site of high affinity of binding ТіО 2 , its composition also includes such amino acids as arginine and lysine, that have the least conformational freedom and a high value of Gibbs free energy. The composition of the fourth site is as following: Hіs34 (+), Arg68 (+), Lys299 (+), Glu302 (-), Lys303 (+), Lys307 (+), Glu332 (-), Lys557 (+), Asp558 (-), Asp559 (-). The binding site under consideration also has non-polar amino acids, whose number regarding the site of comparison increases more than twofold: Gly39, Gly40, Ala53, Gly305, Ala333, Gly334. Glycine with the value of ∆G = 0 kJ/mol, similar to proline (∆G = +3 kJ/mol), causes destabilizing impact on the polypeptide chain, whereas the amino acid alanine does not have any conformational freedom (∆G = -0.77 kJ/mol). As for hydrophobic amino acids, their number does not differ from their content at other binding sites for ТіО 2 . Fig. 8 shows the ratio of content of polar charged amino acids to polar uncharged amino acids in the sites of ТіО 2 binding to the extracellular part of GABA B1а subunit. Thus, the molecular docking of TiO 2 nanoparticle to the extracellular part of GABA B1а of GABA B receptor subunit was performed using PatchDock web-service. The results of these studies demonstrated that TiO 2 in the form of nanosized material is capable of forming a supermolecular structure via interaction with the extracellular part of GABA B1а of GABA B receptor subunit, and it may be assumed that this will be accompanied by modulations in its spatial organization.