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Research paper1

Structural Analysis and Docking of Stilbene2

Synthase Protein from Chinese Grape Vine Vitis3

pseudoreticulata4

5

6Divya K1, Venkata Ramana G1 and K.V.Chaitanya1*7

81Department of Biotechnology, GITAM Institute of Technology, GITAM University, Visakhapatnam-530045, INDIA9

Author’s Contributions10All the three authors have read, write contributed equally for the manuscript11

1213.14ABSTRACT15

16

Aims: The present work aims to perform the molecular modeling of stilbene synthaseprotein from Chinese grape vine Vitis pseudoreticulataPlace and Duration of Study: The study has been performed in the Department ofBiotechnology, GITAM Institute of Technology, GITAM University, Visakhapatnam, INDIA fora period of 8 months.Methodology: The sequence of vitis STS protein was obtained by BLAST search and wassubjected to phylogenetic analysis using UPGMA tree. The secondary, 3D and quarternarystructures have been predicted for the protein and the stability of the structures wasdetermined through Ramachandran plot and PROSA analysisResults: In the tree, Vitis pseudoreticulata STS protein was clustered with Vitis vinifera andthen clustered with Medicago trunculata. The secondary structure of the protein ispossessing helices, coils and sheets respectively and most of the protein is coiled. Twoheterogens CoA and Mg were identified to be omni present in the sequence. Quarternarystructure prediction confirms the predicted structure is thermodynamically stable with thedissociation energy of 16.5 Kcal, which is positive. The predicted model was subjected toevaluation by PROSA with a Z score of -10.1. Ramachandran plot revealed that thepredicted that 96.6% residues were in favoured region, 2.6% were in allowed region and 30.8% were in outlier region proving that the predicted model is acceptable.Conclusion: The structural model of Vitis pseudoreticulata stilbene synthase has beendetermined, which provides an insight of its molecular function towards the understanding ofits importance in the prevention of cardiovascular diseases and carcinogenesis.

17Keywords: Stilbene synthase; Vitis pesudoreticulata, olecular modeling, Phylogenetic18analysis.191. INTRODUCTION20

21Stilbenes are one of the important members in the phytoalexin group of secondary22

metabolites, produced in grapevines, which has numerous remarkable biological properties23

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[1]. Resveratrol (3,5,4’-trihydroxystilbene) is the main representative of this group located in24

the skin of grape berries, involved in the inhibition of the cellular events associated with25

carcinogenesis, neurodegenerative disorders, inflammation, reported to possess antioxidant26

properties, cardio protective properties and is capable of inhibiting the LDL oxidation, during27

the initial stages pathogenesis and atheroscelrosis [2]. Resveratrol shows potent antiviral28

activity against various families of DNA and RNA viruses by inhibiting the growth of vaccinia29

virus [3]. Oxyresveratrol shows inhibitory activity in HSV-1 and in VZV infections [4].30

Resveratrol is synthesized via the well-characterized phenylalanine/polymalonate pathway,31

the key step of which is catalysed by the enzyme stilbene synthase (STS). Stilbene32

synthase belongs to the type III group of the polyketide synthase enzyme super family which33

converts one molecule of p-coumaroyl-CoA and three molecules of malonyl-CoA into 3,4’,5-34

trihydroxystilbene or resveratrol. This enzyme is a dimer with a molecular weight 90 kDa35

having an iso-electric point (pI) of 4.8. A conserved cysteine residue, located in the central36

section of the protein has been shown to be essential for the catalytic activity of STS enzyme37

and represents the binding site for the p-coumaroyl-CoA starting substrate [5].38

The first grapevine STS gene was cloned from Vitis vinifera cv. Optima, and functional39

characterization has been studied in Escherichia coli [6]. Till date, 43 genes encoding STS in40

grapevine have been identified among which 20 of these genes were found to be expressed41

in response to a pathogen attack [7]. Chinese native Vitis pseudoreticulata represent a42

valuable genetic resource for grapevine disease resistance breeding and is a valuable43

resource of STS genes. The structure of the STS protein has not been solved in the vitis44

species. In the present study, we report the molecular structure of the stibene synthase45

protein from Vitis pseudoreticulata and its docking studies to determine the binding abilities46

of this protein, which has also been performed in Silico for identifying the co-factors which47

are interacting with this protein.48

49

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2. MATERIAL AND METHODS5051

2.1. Sequence Information52

The sequences of the stilbene synthase gene from 9 various plants tomato, cotton,53

soyabean, rice, Arabidopsis, grape, Medicago, Ipomea were obtained from DFCI by54

nucleotide BLAST search with Stilbene synthase of Arabidopsis as source. These55

sequences were translated into the amino acid sequences and their ORFs were identified.56

2.2. Phylogenetic Analysis57

A multiple sequence alignment (MSA) of the STS deduced proteins, was performed using58

the clustal W alignment, which takes into account the possibility of large gaps in the59

alignments. A phylogenetic tree was generated with the UPGMA method using MEGA 5.060

software to obtain the tree with bootstrap values at each branch length showing the61

evolutionary relationship between the sequences of input [8].62

632.3. Secondary Structure Prediction64

The secondary structure prediction of the Vitis pseudoreticulata STS protein was carried out65

using protein structure prediction server PSIPRED to identify the similarities among their66

protein structures [9].67

2.4. Ramachandran Plot68

The core of the predicted protein structure or allowed areas in the plot showing the preferred69

regions for psi/phi angle pairs for residues in Vitis pseudoreticulata stibene synthase70

protein was determined through Ramachandran plot using RAMPAGE server [10].71

2.5. 3D Structures, Binding site and Heterogen prediction72

3D Ligand Site is a web server used for the prediction of ligand-binding sites, based on73

successful manual methods used in the eighth round of the critical assessment techniques74

for protein structure prediction.75

2.6. Quarternary Structure Prediction and Model Evaluation76

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PDBePISA is an interactive tool used for the prediction of probable quaternary structures, of77

Vitis pseudoreticulata stilbene synthase protein. The potential errors in the predicted 3D78

models were detected through ProSA (protein structure analysis) tool server. The overall79

quality score calculated by ProSA for a specific input structure is displayed in a plot that80

shows the scores of all experimentally determined protein chains currently available in the81

Protein Data Bank (PDB).82

2.7. Docking Studies of Vitis pseudoreticulata Protein83

The prediction of ligand interactions with Vitis pseudoreticulata stilbene synthase protein84

was performed through Molegro Virtual Docker, which handles all aspects of the docking85

process from preparation of the molecules to determination of the potential binding sites of86

the target protein and prediction of the binding modes of the ligands.87

8889909192

3. RESULTS AND DISCUSSION9394

3.1. Phylogenetic analysis95

Resveratrol is a well-known plant-derived polyphenolic phytoalexin that normally functions as96

an antimicrobial compound in plants, yet also shows significant health promoting effects in97

animal models [11]. Stilbene synthase (STS) is the key enzyme in vitis leading to the98

biosynthesis of resveratrol and stilbenes [12]. In the present study the evolutionay99

relationship of the stilbene synthase gene among different dicotyledenous families with100

respect to the economically important plants was studied by constructing the UPGMA tree101

with boot strap values using MEGA (Figure 1). In the tree, Vitis pseudoreticulata and Vitis102

vinifera (TC 144217) clustered together in a single clade with a branch length of 0.0052,103

which further clustered with Vitis vinifera possessing a branch length difference of104

0.1207.This first cluster then clustered with Medicago trunculata (TC177640) with a branch105

length difference of 0.1374. Segregation of Glycine max (TC 25486) and Gossypium106

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Lycopersicon TC217840

Lycopersicon C217706

Ipomoea TC623

Glycine TC425486

Gossypium TC229867

Arabidopsis TC373951

Arabidopsis TC372306

Glycine TC434240

Medicago TC200217

Gossypium TC260016

Oryza TC488917

Helianthus TC40680

Helianthus TC39714

Vitis TC152941

pseudoreticulata query

Vitis TC144127

Medicago TC177640

Oryza TC494384

Ipomoea TC4048

0.0062

0.0104

0.0104

0.0813

0.1706

0.0363

0.0429

0.0842

0.0999

0.0275

0.0275

0.1666

0.0363

0.0341

0.8115

0.0062

0.0114

0.0429

0.0780

0.0052

0.0518

0.1259

0.00660.0191

0.0341

0.0103

0.0090

0.0082

0.0075

0.0033

0.0029

0.0157

0.0375

0.0292

0.0039

0.6409

hirsutum (TC229867) separately into a cluster with a branch length of 0.0103 is a interesting107

observation. Gycine max (TC 434240) and Medicago trunculata (TC200217) segregated108

separately into a cluster with a branch length of 0.0341 (Figure 1). The two genes of109

Lycopersicon esculentum (TC217840 and TC217706) and Arabidopsis (TC37391 and110

372306) also segregated separately into two different clusters of branch lengths 0.0068 and111

0.518 respectively. The Ipomea (TC623) further clustered to the Lycopersicon esculentum112

(TC217840 and TC217706) cluster with a branch length difference of 0.015. The113

phylogenetic analysis indicates that a very high degree of similarity exists between the Sts114

genes of Vitis pseudoreticulata and Medicago trunculata (Figure 1).115

116

117

118

119

120

121122123124125126127128129130131132133134135136137138

Fig 1. Phylogenetic analysis of stilbene synthase protein By UPGMA Method139140

3.2. Secondary structure of STS protein141

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Proteins are the most complex chemical entities in nature comprising of a large142

number of atoms, variable composition, convoluted topology and complex surface features,143

which make simple descriptions of protein structures almost impossible [13]. Secondary144

structure of a protein will play primordial role in bioinformatics for the drug research and145

development. But the secondary structures of all proteins were not yet predicted. The reason146

is some proteins may be novel proteins or there are no experimentally determined structures147

for the existing sequences to substantiate the requirements and to eliminate this dearth in148

the knowledge. Several databases are providing tools to model the secondary structures149

basing on their Fasta sequences. In the present study, the secondary structure of Vitis150

pseudoreticulata protein was predicted using PSIPRED, which gave clear information151

regarding its protein structure. First helix is from 4 to 9 and the second helix is from 33-44 in152

the protein Similarly in 3,4,8,9,11,12,13 helixes also exhibit subtle differences either in153

inception or culmination of the helixes. Remaining helixes are in same position for both154

structures. In coils 4,5,6,9,12,13,14,16,18,19,22,23,24 and in strands at 2,3,4,6,8,11,12 have155

a one amino acid residue difference either in inception or culmination (Figure 2 and Table1).156

Table 1. The amino acid distribution of Vitis pseudoreticulata secondary structure157158159

Structure Aminoacids160161

Coil 1-3,10-16,26-32,45-49,61-64,70-73,78-90,117-125,131-139,148-165,181-162184,193-205,215-219,225-237,241-244,247-256, 260-261,268-270,290-163298,303-307,317-320, 332-339,353-365,385-391164

165Helix 4-9,33-44,50-60,74-77,91-116,140-147,166-180,206-214,271-289,308-166

316,321-331,340-352,374-384167168

Strand 17-25,65-69,126-130,185-192,220-224,238-240,245-246,257-259,262-169267,299-302,366-373170

171

172

173

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Fig.2. Secondary structure of Vitis STS protein174

175

176

177178179180181182

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3.3. 3D Structures, Binding site and Heterogen prediction183

Analysis of the 3D structures is much easier when compared to the analysis of binding site184

prediction or heterogen prediction. It was powered by jmol view with easy color185

differentiation between various heterogens and unique color expression of binding sites186

enhancing easy identification in complex structures of Vitis pseudoreticulata (Figure 3 and187

Table 2). Binding sites indicted with a blue color along with labeling. Heterogens COA and188

Mg were differentiated in multiple colors. Our prediction of binding sites revealed that the189

heterogens COA and MG are omnipresent in the sequence. 57th amino acid residue with190

contact points 10 of Vitis pseudoreticulata stilbene synthase protein has the lowest JS191

divergence of 0.60 with the average distance of 0.09. The highest JS divergence of 0.91 was192

found at 164 residue with a contact point 8 and average distance 0.31. If the space fill format193

activated, it will be more feasible to distinguish between metal ions and binding sites. The 3D194

Structures with the labeling and JS divergence are shown as screen shots (Figure 3 and195

Table 2).196

Fig.3.3D Structure Prediction of Vitis pseudoreticulata stilbene synthase protein197198

199

200

201

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Table 2. Binding site prediction of Vitis pseudoreticulata stilbene synthase protein202

structure203

204205206207208209210211212213214215216217218219220221222223224225226227228229230231232233234235236237238239240241242243244245

246

247

248

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249

250

251

252

253

3.4. Quaternary Structure prediction254

Quaternary structure plays an important role in defining the function of a protein by255

facilitating allostery and co-operativity for the regulation of ligand binding[15]. In the present256

study the quaternary structure of the Vitis Stilbene synthase protein was predicted using257

PISA by considering the best assembly which comprises maximum structure size with good258

stability (Table 3). Our result confirms that the predicted STS protein structure is259

thermodynamically stable with 16.5 dissociation free energy which is positive. ΔGint value is260

-15.3 Kcal/M indicating that the dissociation energy is less than the association energy of261

this assembly (Table 3).262

Table 3. Quarternary structure prediction of Vitis pseudoreticulata stilbene synthase263protein264

265PQS mm Size Stability Surface Area Buried Area ΔGint ΔGdiss266

Sq A Sq A Kcal/mol Kcal/mol267268

1 2 Stable 63230 5620 -15.3 16.5269270

2713.4. Model evaluation by PROSA272

ProSA-web z-scores of all protein chains in PDB were determined by X-ray crystallography273

(light blue) or NMR spectroscopy (dark blue) with respect to their length. The plot shows only274

chains with less than 1000 residues and a z-score ≤ 10. The z-score of Vitis275

pseudoreticulata was highlighted as large black dots in the light blue region representing that276

the structures are X-ray crystallography structures with a Z score value of -10.1 (Figure 4). Z277

score plots elucidated that structure of Vitis pseudoreticulata possess model quality [16]. In278

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this plot, sequence position was plotted against knowledge-based energy. Window 10 and279

40 indicated in the inset represents number of residues considered for plotting their average280

energies. Window 10 graphs with more fluctuations given trivial importance hence shaded in281

light color. To the mean for 40 residues in Vitis pseudoreticulata the average energy was282

greater than -2 with reference to knowledge based energy predictions. Thin lined graphs in283

window 10 with more fluctuations ranging between -3 to 1 showing higher energies than284

windows 40 graphs.285

286

287Fig.4. PROSA (Protein Structure Analysis)-Z Plot of Vitis pseudoreticulata stilbene288synthase protein structure289

2903.5. Ramachandran Plot Analysis291

The Ramachandran plot shows the phi-psi torsion angles for all residues in the structure292

except those at the chain termini. Glycine residues are separately identified by triangles as293

these are not restricted to the regions of the plot appropriate to the other side chain types.294

The colouring/shading on the plot represents the different regions were as described by [14].295

The darkest areas correspond to the "core" regions representing the most favourable296

combinations of phi-psi values. The percentage of residues in the "core" regions is one of297

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the better guides to the stereochemical quality. The Ramachandran plot for Vitis298

pseudoreticulata using RAMPAGE, revealed that among the 386 residues, 373 (96.6%)299

were in favoured region, 10 (2.6%) were in allowed region and 3 (0.8%) were in outlier300

region proving again that the predicted model is acceptable (Figure 5). Ramachandran plot301

for general, glycine, pre-proline and proline were also performed and it showed the glycine,302

pre-Pro and proline of Vitis pseudoreticulata falling under allowed regions and also those303

glycine residues falling in disallowed region (Figure 5). The overall results provided in the304

study confirm that the predicted 3-Dimenrsional structure of Vitis pseudoreticulata is305

acceptable and of good quality.306

307308309310311312313314315316317318319320321322323324325326327328329330331332333334335336337338339340341

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342343344345

Figure 5. Ramachandran Plot of Vitis pseudoreticulata stilbene synthase protein346secondary structure347

3483.5. Docking of Vitis pseudoreticulata STS Protein349

Protein docking is molecular modeling technique, which predicts the position and orientation of350

ligand when it is bound to the specific protein or its receptor, which has a wide amount of351

applications in the pharmacy for identification of specific target [17]. In the present study, the352

stilbene synthase protein of Vitis pseudoreticulata was docked with the COA ligands (Figure 6).353

The docking of protein was set up with 15 COA ligands for 10 runs. The whole time taken for354

docking was 3 hour 45 minutes at the end of which COA_392 [B] showed the lowest energy with355

a Moldock score [grid] of -177.858 COA_392 [B] is the best ligand (Figure 6). The energy of the356

best ligand was given graphically as a blue line graph. The amino acid residues showed docking357

at 58-63,98,101,104-109,131,161,162,165,192,207-215,217,218,253,268-73,271,272,306-358

309,275,311, 329,330,333,334,343-345,347-352 respectively (Table 4). The docking pattern at359

58-63,207-215,271,272,306-309 amino acids was similar to the predicted ligand sites by 3D360

Ligand site was an interesting observation made in this study.361

362

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363364365

Fig. 6. Structural view of Vitis pseudoreticulata stilbene synthase docking366

Table 4 Docking Sites of Vitis pseudoreticulata stilbene synthase protein367368

Amino Acids Details369370

58-63,76,98,101,104-109,131,161,162,165, Docked371192,207-215,217,218,253,268,271,272,273,372275306-309,311,329,330,333,334,343-345,373347-352374

375164,206,254,267,305 has cavity but not docked37658-63,207-215,271,272,306-309 amino acids was similar to the predicted ligand sites377

378379380

4. CONCLUSION381382

The structural analysis of proteins is important for understanding its function based on their383

positioning of specific amino acids at target sites. In the present study, the structural model384

of Vitis pseudoreticulata stilbene synthase has been determined, which provides an insight385

of its molecular function towards the understanding of its importance in the prevention of386

cardiovascular diseases and carcinogenesis. Further studies are in progress for comparative387

analysis and homology modeling of STS protein from medicinal plants.388

389

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ACKNOWLEDGEMENTS390391

The research in the lab of KVC is supported by the funding of DST and DBT, Govt. of India.392

VRG acknowledges DST for the Research Fellowship.393

394AUTHORS’ CONTRIBUTIONS395

396All authors have contributed equally for the manuscript.397

398399

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