Homology modeling of functional proteins of smilax aspera plant and

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Bikash and Samrat, 2015. International J Ext Res. 5:72-78http://www.journalijer.com

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Homology Modeling of Functional Proteins of Smilax aspera Plant and Its Docking Study with p53 Protein

Bikash Thakuria and Samrat Adhikari*Department of Biotechnology

St. Edmund’s College, Shillong 793003 India

AbstractSecondary metabolites from Smilax aspera plant, also known as Sarsaparilla possesses vital proteins which are capable of treating various ailments and are of great medicinal value. Interestingly this plant has been less exploited for medicinal properties and hence the present study is based on the in silico approach to characterize the important functional proteins and its role in in-hibiting the proliferation of the p53 protein during cancer cells proliferation. Twenty two important functional proteins from this plant have been reported but only five proteins were selected due to the availability of their complete sequences. These five sequences were further explored for the putative domain content, homology modeling, computation of the physiochemical prop-erties and finally docking analysis with the p53 protein using PATCHDOCK server. The results suggest that among the five pro-teins, Ribulose 1,5-bisphosphate carboxylase oxygenase with the template of 1WDD has the highest docking score followed by the other proteins from this plants. The analysis further reveals that these structural important functional proteins may probably be engineered for developing suitable agents for anti-cancer therapy.

Keywords: Homology, protein motifs, proteins, structures and databases.

*Corresponding author e-mail: [email protected]

IntroductionBioactive compounds exploited from the plant metabolites, have been a centre of attraction for research in the present decade against cancer therapy because of its lesser toxic nature than the usual chemotherapeutic drug molecules (Somasagara et al. 2012). There have been enormous reports on the phytochemicals pres-ence in fruits and vegetables which are more effective than their individual constituents in preventing cancer through both addi-tive and synergetic effects (Eid et al. 2012). Many medicinal plants indeed in different parts of the world have been characterised to have anti-cancer properties in their secondary metabolites and extensive studies have been carried out so far regarding treat-ment against cancer and other diseases (Rafatullah et al. 1991). In context to the present work Smilax aspera has been found to have numerous medicinal values with endless curation for disease

biology. The phytochemicals extract (viz. cyanidin 3-o-rutinoside, pelagonidin 3-O-rutinoside, steroidal saponins and safrole) pos-sesses the properties of inflammation modulating and anti-rheu-matoid activity and has helped a number of patients across the globe (Russo et al. 2012). Beside the above mentioned definitive applicability, these plant extracts have also been involved in curing gout, diabetes, fibrocystic and bacterial diseases, aging and neuro-logical disorders (Salini et al. 2012). Furthermore the presence of carotenoids in plant extract attracts them as a potential agent for exploitation for drug designing in treating cancer and also various other diseases. Lycopene was found to be the main carotenoid in the pulp, followed by β-carotene and β-cryptoxanthin (both free and esterified forms). Other minor carotenoids reported were ly-cophyll , zeaxanthin, lutein, and antheraxanthin (Ruano-Ravina et al. 2000; Cook et al. 1999). Anthocyanins pigments extracted from the berries of Smilax aspera plant, a creeping shrub of the Med-iterranean region have shown presence of pelargonidin 3-O-ruti-noside and cyanidin 3-O-rutinoside which has wide medicinal properties (Longo and Vasapollo, 2006). In India Smilax aspera is

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widely distributed in the North-Eastern part of Brahmaputra val-ley along the foot hills of Assam Hills (Tiwari, 2008). Owing to the significant medicinal value of this plant 14 primers being reported with the help of a dual-suppression-PCR method and 22 proteins have been identified but the exact functions & annotations have not been explored in detail (Ivanova et al. 2011). Therefore based on the sequences of these proteins the present work has been con-ceptualized at the molecular level using appropriate in silico tools with a mandate objective for the future prospects to explore the biological functions of these proteins in experimental analysis. The bioinformatics approach using appropriate algorithms has enormously enhanced the outlook of the medicinal aspects of the various plant metabolites. The algorithms in turn depend on theoretical foundations such as discrete mathematics, control the-ory, system theory, information theory and statistics. Automated prediction of the protein-protein interactions and protein-small molecule is one of the most challenging problems in structural biology (Lv et al. 2013; Promal et al. 2013). In the present work the 22 proteins reported for the Smilax aspera were subjected to homology modeling for selection of the best proteins and docking analysis were performed with the p53 protein to enunciate its role as anti-cancer properties.

Materials and MethodsRetrieval of sequences and BLASTThe sequences of the 22 functionally important proteins of Smi-lax aspera have been retrieved from the NCBI databases with Accession Nos. ADF29634, AEN85477, ADF29634, ADF29645, AEN85479, ADF29644, ADF29644, ADF29642, AEK34583, AEQ48877, AEQ45578, AEQ45576, AEN85478, CAA58951, AEM05279, ADF29641, AEM05281, ADF29635, AEK34458, AEM05280, CCG28501 and ACB88375. These 22 proteins se-quences were used for the BLAST analysis and appropriate hits were selected. The tumour suppressor protein sequence p53 has been retrieved from the PDB database with template 1TUP (Cho et al. 1994).

Domain analysis The sequence selected from the BLAST hits were analysed for Do-main architecture using NCBI domain database CDART (Geer et al., 2002) and functional domain were computed with motif scan (Naughton et al. 2006).

Physiochemical AnalysisThe physiochemical characterization like theoretical isoelectric

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point (pI), molecular wt, no. of positively and negatively charged residues, extinction coefficient, instability index, aliphatic index and Gradient Average Hydropathicity (GRAVY) were evaluated using the ExPASy ProtParam server (Gasteiger et al. 1999). Homology Modeling and structure validation Homology modeling for the functionally important proteins was performed using CPH model server (Neilsen et al. 2010) with tem-

plates 1WDD, 1WWL, 2JUO, 3BBN and 3 LUO respectively. Eval-uation of the 3D structures modeled was carried out using MOL-PROBITY (Chen et al. 2010). The constructed models were energy minimized by QMEAN server (Benkert et al. 2009) and structures were visualized by PYMOL software tools.

Molecular DockingDocking study of the homology modelled proteins was carried

Table 1. Physiochemical parameters of the 5 accessory proteins of the Smilax aspera plant. The isoelectric point, molecular wt, negatively charged amino acids, positively charged amino acids, extinction coefficient, instability index, aliphatic index, estimated half-life and GRA-

VY are described in separate columns

Protein with Swis-sProt entry name and the no. of amino acids

Mol. Wt. Theoretical pI

Negatively charged residues

Positively charged residues

No. of atoms

present

Estimated half-life (with respect to E.

coli)

Instability index

Aliphatic index GRAVY

1WDD (D5LM-K1)446aa 49458.2 6.34 55 50 6899 2 minutes 38.4 78.99 -0.272

2JUO (G3F7W1) 416aa 49288 9.54 26 46 6967 >10 hours 53.15 98.37 -0.085

1WLL (D5LMJ4) 408aa 48280.5 9.34 27 41 6796 >10 hours 51.15 95.51 -0.119

3BBN (Q9TJV2) 196aa 22707.6 10.7 16 39 3279 >10 hours 49.78 100.46 -0.556

3LUO (B2LLV5) 171aa 18898.3 9.3 14 19 2712 3 minutes 36.19 114.04 0.178


out using PATCHDOCK tools (Laurie & Jackson, 2005) and en-ergy minimization was performed before and after docking using QMEAN server (Benkert et al. 2009).

Results and DiscussionThe BLAST results predicted regions of local similarity between sequences. The program compares nucleotide or protein sequenc-es to sequence databases and calculates the statistical significance of matches. Therefore in the present study, BLAST analysis of the 22 protein sequences (both complete and partial) retrieved from the NCBI databases was performed. Out of these 22 protein se-quences only 5 proteins were selected as their complete sequence were available. These 5 sequences were further evaluated for the characterization using bioinformatics tools. The sequences were further analysed for the presence of putative functional domains using CDART tool and the results depicted as positive aspects and consequently enunciated a picture for similarity score with the presence of protein superfamily in resemblance with the other or-ganism (Figure 1). Domain architecture of Maturase K proteins showed varied amino acid sub unit each comprising of 408aa and 416 aa residues. These two subunits of Maturase K consequently showed the presence of one common domain of Intron_maturas2 superfamily which basic function is intron-encoded reverse tran-scriptase activity and also DNA endonuclease activities. The other domains present were Ribosomal S4 superfamily/S4 of Ribosomal protein S4 with subunit of 196aa and RuBisCo_large for the protein

Ribulose 1, 5-bisphosphate carboxylase oxygenase with subunit of 446aa. The 196 subunit domain has a wider function to bind to ribosomal RNA/ domain and the 446 aa protein basically catalyzes the primary CO2 fixation step in Pentose Phosphate Pathway. The analysis also depicted the presence of another domain belonging to RNAP N superfamily comprising of 171 aa subunit of RNA polymerase C protein. Furthermore all the subunit of the 5 pre-dominant proteins taken into consideration for the present study also revealed that their unique putative domains range in various organisms from smaller eukaryotes to higher organism which are shown in Fig. 1. Motif scan analysis of the proteins suggested the nonexistence of various functional domains which has to be fur-ther explored due to absence of competent crystal structure.

The physiochemical properties like theoretical isoelectric point (pI), molecular wt, no. of positively and negatively charged resi-dues, extinction coefficient, instability index, aliphatic index and Gram Average Hydropathicity (GRAVY) of the proteins were evaluated with ProtParam analysis (Table 1) and based on the parameters they exhibit the structures were further subjected to homology modeling. The homology modeling performed using CPH modeling software revealed generation of 5 templates name-ly 1WWL for Maturase K, 1WDD for Ribulose 1, 5-bisphosphate carboxylase oxygenase, 2JUO for Maturase K, 3BBN for ribosom-al protein S4 and 3LUO for RNA pol. C. The structures of these proteins obtained by homology modelling generated alpha helices, beta strands and loops which are shown in Fig. 2. Comparatively the amino acid percentage in the favourable region in the CPH

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models were found to be 91.2 % for 1WDD, 77.7 % for 1WWL, 78.5% for 2JUO, 81.7 % for 3BBN and 89.8 % for 3 LUO whereas the amino acids percentage in the disallowed region were com-puted to be 0.2 %, 1.3 %, and 1% for 1WDD , 2JUO and 3BBN respectively. The amino acids percentage in the disallowed region for 1WWL and 3LUO were found to be zero which suggest that the protein structure is intact as well. The data allocated using CPH models also suggested that the proteins has the highest homology percentage and also showed the highest percentage of amino acids in the allowed region.

The CPH modelled structures were further validated by Ram-achandran Plot. Ramachandran plot revealed that 3 amino acids Val (331), Lys (70), Glu (33) for 1WDD, 2 amino acids Ala (29), Glu (12) for 1WWL, 8 amino acids Thr (15), Arg (120), Asp (31), Lys, (12), Glu (16), Phe (133), Val (131), Leu (102) for 3BBN, 2 amino acids Asp (72), Thr (116) for 2JUO which basically showed presence on the 4th quadrant suggesting that the Ramachandran plot has validated the structure obtained from CPH modeling. The model also studied the superimposition of the active sites and RMS values using the DALI superimposition tools. The superim-position of the structures generated by CPH modeling revealed the conformational changes and it directs to a mechanism for ac-commodating substrates of different sizes. The RMS value for all the proteins containing putative domains was computed out to be 3.7 which were statistically significant.

In recent years a number of spectacular discoveries on surpris-ingly similar structures of proteins whose evolutionary kingship could not be recognized alone based on primary structure (Gibrat et al. 1996). Hence secondary structures allow a simple and intu-itive description of 3D structures which are widely employed in a number of structural studies. Thus the application of secondary structure prediction basically aids in classifying proteins and as-sisting in separating domains and identifying particular function-al motifs. Based on the current literature regarding 3D structures and the amino acids sequence data, it appears that these functional proteins from Smilax aspera share a common domain with many other related eukaryotes and further raised a question whether these protein have some other biological functions.

The p53 protein family regulates many vital biological processes including cell differentiation, proliferation, cell cycle checkpoints and also apoptosis. As such, p53 has been described as “the guard-ian of the genome” because of its role in conserving stability by preventing genome mutation. The abnormal expression of this protein contributes to carcinogenesis (Levine, 1997). Computa-

tional docking analysis using protein interactions has emerged a powerful tool for evaluating consensus formulation for inhibit-ing the proliferation of p53 protein in recent years (Ai et al. 2013 and Carper et al. 2014). Literature on the exploitation of phenolic compounds, phytochemicals, chemical based drugs etc. for dock-ing interaction with p53 protein has been extensively reported and has generated wider applications for various drug designing tools (Aloy & Russel, 2006). In the present study a flexible body docking analysis mode was followed using PATCHDOCK server of p53 molecule with the homology modelled 5 proteins of Smilax aspera and the data revealed significant interactions of geometry based algorithm and the computed Global Energy, Docking Score, attractive VdW, repulsive VdW, ACE and H bonds parameter are depicted in Table 2 and the docking interactions with the p53 mol-ecule are shown in Fig. 3. The result of docking analysis suggested that the template of 1WDD of Ribulose 1,5-bisphosphate carboxy-lase oxygenase showed the highest docking scores followed by the other templates which were comparatively low. The global ener-gy of the 1WDD template also confirms the same. The template of 3LUO showed lowest docking score as probably of the reason that without having either of H donor or acceptor bonds were un-able to fit in the active sites pockets of the p53 (1TUP) and lack any structural interactions with the enzymes through H bonds respectively. The docking results of 3BBN could not be evaluat-ed because of the enormous complexity of the protein structure. Plants showing the presence of vehement metabolites in them has fetch attention of many medicinal approaches towards them. In reference to the present study the proteins from Smilax aspera which has its enormous medicinal values in almost all the spheres of curing several diseases including cancer has to be the focussed and the docking parameters evaluated as to be further validated by experimental procedures. Furthermore with the help of Bioin-formatics tools available the present work only could predict the suitable protein-protein interactions but the ligands associated with them have to be emphasized and evaluated using suitable ex-perimental procedures. Henceforth, the in silico approach towards any medicinal aspects of plant extracts in curing any disease would pave the pathway to follow and achieve it and easy availability of these plants would make it more convenient to use against various diseases. Smilax aspera, with the functional proteins present in it opens up a new dimension in the field of medicines. The differ-ent proteins present in it and the corresponding Ramachandran Plots associated with the proteins gives us the idea of the correct measures to implicate and design strategy towards the drug de-

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Receptor -Ligand Global Energy Docking Score Attractive VdW Repulsive VdW ACE HB

1TUP 1WDD 40.54 23408 -15.71 48.73 3.5 -0.33

1WWL 13.06 19922 -2.33 0.01 0.92 -2.29

2JUO 8.29 20588 -2.56 2.1 1.16 -0.46

3BBN - 18216 - - - -

3LUO 6.8 19184 -0.99 0 0.2 0

Table 2. Docking Scores and Global energy parameters of protein docking interaction of the functional proteins of Smilax aspera with p53 protein. The different parameters such as attractive Van der Waals (VdW), repulsive Van der Waals (VdW), Atomic Contact

Energy (ACE) and Hydrogen bonds (HB) are computed for the each of the proteins


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Conflict of interestsThe authors hereby declare that they have no conflict of interest regarding the paper.

AcknowledgementsWe take this opportunity to acknowledge the funding received from the Department of Biotechnology, Govt. of India for setting up Bioinformatics Centre under BTISNET programme at Depart-ment of Biotechnology, St. Edmund’s College, Shillong. We also express our heartfelt gratitude to Dr Sylvanus Lamare, Principal, St. Edmund’s College, Shillong for his support & encouragement throughout the work.

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signing methodology for the welfare of mankind in near future. Apart from the above mentioned approach the plant extracts can also be in reach of the people residing in the local areas where it grows and give them a traditional boost in the application of the extracts in curing diseases in a natural way rather than the costlier chemotherapeutic and radiation technology involved.

ConclusionsFrom the present study on the molecular characterization of the Smilax aspera accessory proteins, Ribulose 1,5-bisphosphate car-boxylase oxygenase with the template of 1WDD has the highest docking score followed by the other proteins. Further it might be concluded that using bioinformatics tools it could be possible for exploration of this plant for the anti-cancer property which in-deed would require a substantial experimental methods.

Article Information:Received: 01 May 2015 Accepted: 25 May 2015Online published: 26 May 2015

Cite this article as:T Bikash and A Samrat, 2015. Homology modeling of function-al proteins of Smilax aspera plant and its docking study with p53 protein. International Journal of Extensive Research. 5: 72-78.


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