DOCKING STUDIES OF STRESS TOLERANT PROTEINS WITH PROTECTIVE MOLECULES

: The present study was aimed to dock the stress tolerant proteins with the protective molecules. The stress tolerant proteins namely hexokinase, citrate synthase, phosphofructokinase from Selaginella moellendorffii, transketolase, transketolase 7 from Craterostigma plantagineum which can interact with the protective molecules trehalose sugar and growth regulating compounds IAA and tryptophan. The docking studied was performed by using the docking tool GOLD. The active sites were predicted using Q-site finder. The results were analyzed based on the binding compatibility of H bonds and the interactions energy was mentioned as k.cal/mol. Keywords : The results were analyzed based on the binding compatibility of H bonds and the interactions energy was mentioned as k.cal/mol.


INTRODUCTION
Carbohydrate plays a very important role to protect the cells due to the accumulation of sugars; their abundance was dramatically changed during dehydration and rehydration [1] . The disaccharide stress response to sugar trehalose plays a key role as osmoprotectant to protect cells, leaves, protein, enzymes from drought [2][3]. Rosa and James [4], Gabriel et al., [5] studied that trehalose as the molecular chaperon protects the citrate synthase from dehydration. During desiccation the trehalose sugar perform stabilization reflects that the substitution for solvent water in H bonding to proteins and prevent lipid transition at membrane, and prevent the aggregation of denatured proteins and form a glass like structure around the proteins [6]. The plant growth regulator IAA present in all plants as free forms or sometimes it can bind with proteins and amino acids, when the auxin bind with the proteins it makes more rigid and stabilized the molecules [7] . IAA concentrations highly occurred during desiccation in Craterostigma wilmsii [8]. Auxin protects the plants from various abiotic stresses [9 -12]. The auxin synthesis and transport offered the plant to adapt various stress [13]. Amino acids deposition has been observed in many plants exposed to abiotic stress [14]. Citrate synthase is an enzyme responsible for the catalyzing the reaction of the citric acid cycle the condensation of acetyl-coA and oxaloacetate to form citrate [15]. John et al., [16] observed the trehalose mediated high level of protection to the phosphofructokinase during dehydration. Phosphofructokinase involved in the regulation of glycolysis in animals [17], plants [18], insects [19], bacteria [20] and yeast [21]. Hexokinase is an enzyme converts glucose into glucose 6-phosphate by phosphorylation. Hexokinase inhibited by its own product of glucose-6-phosphate glucose [22]. During dehydration the hexokinase activity linked to stress response sugar accumulation lead to decrease the glucose concentration at the desiccated state of Sporobolus stapfianus and X. viscosa [23]. Transketolase involved two reactions in pentose phosphate pathway, converts ribulose 5-phosphate and xylulose 5-phosphate to glyceraldehydes' 3 phosphate and sedoheptulose 7phosphate and also it converts xylulose 5-phosphate and erythrose 4 -phosphate to form glyceraldehyde 3-phosphate and fructose phosphate. Docking is the interaction process between the orientation of target protein and ligands molecule to produce the stable compound [24].
Orientation offered the strength of association or binding affinity between the target protein and ligands molecules [24]. At present number of docking tools are available in the market. In the present study the GOLD (Genetic Optimization of Ligand Docking) based on genetic algorithm (GA) was employed for the docking of the plant compounds. Deepika et al., [25] confirmed that hydrogen bonding interactions play an important role for stability of the complex. Feruloyl CoA was docked with Cinnamoyl CoA reductase (CCR) of Leucaena leucocephala using GOLD [26]. Similarly the Aeromonas hydrophila efflux B protein was docked with (EPIs) 1-(1naphthylmethyl)-piperazine (NMP) and phenyl-arginine-βnaphthylamide (PAβN) [27] and STAT4 protein with Flurbiprofen [25]. Docking study was carried out IAA with lectin protein obtained from Canavalia maritima seeds [28]. With this knowledge the present study was aimed dock the following proteins namely hexokinase, citrate synthase, phosphofructokinase from Selaginella moellendorffii, transketolase, transketolase 7 from Craterostigma plantagineum with trehalose sugar, IAA and tryptophan has been studied using the dock tool GOLD.

Materials and Methods
GOLD (Genetic Optimization of Ligand Docking) GOLD score can be calculated Gold score = S (hb_ext) + S (vdw_ext) + S (hb_int) + S (vdw_int) Where S (hb -ext) is the protein-ligand hydrogen bond score, S (vdw -ext) is the protein-ligand van der Waals score, S (hb-int) is the score from intermolecular hydrogen bond in the ligand and S (vdw-int) is the score from intermolecular strain in the ligand.

Ligands (trehalose, IAA and tyrptophan) for binding
The ligands selected for this study were trehalose, indole acetic acid and tryptophan " Fig. 1, 2, 3". The interaction of trehalose (sugar joined by 1-1 alpha bond α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside. This bond makes the trehalose stable to high temperature and resist to acid hydrolysis. Trehalose form a clustering tendency due to the hydrogen bond formed between one another. This association in water to form a cluster in various sizes [29]. The ring structure Tryptophan is the precursors of IAA, IAA (indole acetic acid) or auxins are compounds with an aromatic ring and a carboxylic acid group, it generates majority of effects in plants. The 2D structure of Trehalose was drawn using ACD chemsketch (www.acdlabs.com).  [30]. GOLD software based on genetic algorithms (GA) which expose the ligand conformational flexibility and the rational flexibility of selected receptors hydrogen. Grid was drawn for the selected proteins with the center and the size of bounding box 10 Å. The coordination of the closing box (x=121 Å; y = 87 Å z =45 Å) were characterized from the initial set of active site residues.

Molecular docking
Docking of trehalose sugar with citrate synthase, phosphofructokinase, hexokinase, transketolase and transketolase7 was carried out using GOLD software. The result of docking exposed the best fits of trehalose IAA and tryptophan with the binding receptors of proteins The results were analyzed based on the binding compatibility. Docking energy mentioned as k.cal/mol. 57 amino acids were active site for citrate synthase, 87 amino acids were active site for phosphofructokinase, 64 amino acids were active site for hexokinase, 80 amino acids active site for transketolase and 38 amino acids were active site for transketolase7 were observed. The docking results of citrate synthase with trehalose six H bonds with the score 54.78 k.cal/mol " Fig. 4", three H bonds with IAA with the scores of 43.8 k.cal/mol " Fig. 5", five H bonds with tryptophan with the scores of 49.63 k.cal/mol " Fig.6".      Table -1. High number of hydrogen bond interactions, it is supposed to be most active residue in interacting complex. Trehalose exhibits the highest Gold fitness with citrate synthase score of 54.78 and interacts with six hydrogen bonds and the best docking conformation showed residues SER, MET, ILE, GLY, THR. This kind of interaction may help to stabilize protein during desiccation.

DISCUSSION
The proteins of citrate synthase, phosphofructokinase, hexokinase, transketolase and transketolase 7 were used for the docking studies with desiccation tolerant sugar trehalose, IAA and tryptophan. Trehalose is the essential components of the stress adaptive mechanisms [4]. Trehalose protects the macro molecules during desiccation [31]. Auxin regulates plant defence response and growth and structures [32][33][34]. When IAA interact with sugars mono and disaccharides produced low molecular compounds, as the result of IAA binds with proteins or polysaccharides produced high molecular weight compounds [35]. Tryptophan and IAA possess low binding affinity with lectin proteins Canavalia maritima seeds [29]. Branimir [7] studied the auxin binding protein and molecular modelling of auxin and antiauxin proteins. Nan [36] studied the interaction of auxin with S-adenosyl-L-homo-Cys (SAH). The interaction of sugars with citrate synthase involved the thermal protection was proved through in vitro studies [4]. The docking results explored the H bonding interaction of trehalose with citrate synthase and phosphofructokinase. Some of the proteins are classified as desiccation tolerance they worked both in dehydration and rehydration condition. Some of the proteins play a role of plant stress adaptations [37]. Stress induced proteins play a role to protect cellular damage involved in antioxidant defence, deposition of sugars, cell wall protection and remodelling [1]. Dehydration and heat stable enzymes occurred in plants throughout desiccation and also involved in carbohydrate metabolism, transcription factors and regulatory molecules [38][39][40]. Hexokinase is also a heat stable enzyme involved in carbohydrate metabolism during dehydration. Hexokinase is the very important enzyme which can be worked both in dehydration and rehydration, in desiccated state which converts all the simple sugars to stress response sugars sucrose during desiccation [23]. IAA one of the hormones which control stresses and lead to fight against defence response and plant growth response [41]. Transketolase are involved in reductive and oxidative pentose phosphate pathways and its isoforms transketolase 7 responses to synthesis octulose [42]. The genes of transketolase were identified in resurrection plant C. plantagineum [43]. Wang et al., [44] studied the protein transketolase response in stress tolerance in S. lepidophylla and the enzyme concentration was increased during dehydration of P. patens. Sugars protect protein through the H bonding to the polar amino acids in the protein [45], [46]. During interaction of sugars with phospholipids in the dried state can replace water molecules around the polar head groups [47]. Trehalose acts as the effective stabilizing molecules to protect the biological molecules against desiccation [48], [49]. Trehalose possesses unique interactions with biological systems in the dried condition which is based on water replacement hypothesis [46]. The low concentration of sugar cause to break water-water hydrogen bond and promote sugar water hydrogen bonds. The increase concentration above 25-30% of trehalose cause to increase water-water interactions lead to cause more stable and widely hydrogen bonded water clusters, these concentrations of sugars called as structure makers [50]. Trehalose, mannitol and lactose these combined sugars can preserve some enzyme proteins namely lactate dehydrogenase or phosphofructokinase through water replacements [51]. The present study revealed the interactions of stress tolerant proteins with trehalose sugar and these three stress tolerant enzymes possess weak interaction with trehalose sugar which was proven by inslico method using GOLD software.

Conclusion
The selected proteins were undergoes docking with bio molecules viz trehalose, IAA, and tryptophan through H bonds by GOLD software. Out of 10 results, the best one was selected based on the high score. In the docking process hydrogen bonds play a major role in structure and function of biomolecules.