Homology Modeling of Phosphoenolpyruvate...

Journal of Pharmacy Research Vol.5 Issue 2.February 2012

Akalesh Kumar Verma et al. / Journal of Pharmacy Research 2012,5(2),1248-1255

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Research ArticleISSN: 0974-6943

Available online throughhttp://jprsolutions.info

*Corresponding author. Jashodeb Arjun,Asst. Prof. Department of Zoology,Lumding College,Lumding-782447,Assam, India.

INTRODUCTION

Homology Modeling of Phosphoenolpyruvate Carboxykinase of Ascaris suumAkalesh Kumar Verma1, Ananta Swargiary2, Surya Bali Prasad1 and Jashodeb Arjun*3

1 Cell and Tumor Biology Laboratory, Department of Zoology, North-Eastern Hill University, Shillong, India.2 Parasitology Laboratory, Department of Zoology, North-Eastern Hill University, Shillong, India.

3 Department of Zoology, Biochemistry Division, Lumding College, Lumding-782447, Assam, India.

Received on:10-11-2011; Revised on: 15-12-2011; Accepted on:12-01-2012

ABSTRACTPhosphoenolpyruvate carboxykinase (PEPCK) is present in parasites as well as in their mammalian hosts it serves different functions. In the hostPEPCK catalyses the reaction from oxaloacetate to make phosphoenol pyruvate and carbon dioxide, part of the gluconeogenesis pathway. ParasitePEPCK catalyses the reverse reaction to form oxaloacetate, the precursor of malate, which is metabolized anaerobically by the mitochondria. Thisfunctional difference is related to significant differences in the molecular properties of the parasite PEPCK. These differences between the mammalian andthe parasite enzyme support the belief that PEPCK should be further investigated as a possible target for selective chemotherapeutic agents. The 3Dstructure of PEPCK of any helminthes group is not available in protein data bank. Therefore, based on the knowledge of the template, a three-dimensionalmodel of PEPCK of Ascaris suum was predicted using Modeller 9 v 10 software and processed in to energy minimization, Ramachandran plot analysis,quality assessment and finally deposited into Protein Model Database. Infections with parasitic helminthes are important causes of morbidity andmortality worldwide. New drugs that are parasite specific and minimally toxic to the host are needed to counter these infections effectively. Therefore,the present study will help to target this enzyme for further development of new antihelminthic drugs.

Key words: Phosphoenolpyruvate carboxykinase, Ascaris suum, antihelminthic, parasite, enzyme

Helminthiasis is a crucial problem worldwide, because it is not only a majorcause of human morbidity in the tropics as well as temperate climates [1], butis responsible also for enormous economic losses in livestock animals. Re-cent global estimates indicate that more than a quarter of the world’s popu-lation are infected with one or more of the most common of these parasites-the roundworm, Ascaris lumbricoides; the hookworms, Necator americanusand Ancylostoma duodenale; and the whipworm, Trichuris trichiura [2].Furthermore, the emergence of resistance against generally used anthelminticsmakes the problem more serious [3]. Therefore, the development of noveland potent anthelmintics is urgent.

Helminths have exploited a variety of energy transducing systems in theiradaptation to the peculiar habitats in their hosts, differences in energymetabolisms between the host and helminths are attractive therapeutic tar-gets of helminthiasis. NADH-fumarate reductase is part of a unique respi-ratory system in parasitic helminths [4–7] and is the terminal step of thephosphoenolpyruvate carboxykinase-succinate pathway, which is foundin many anaerobic organisms [8–10]. The composition and linear sequentialorder of the respiratory components of NADH-fumarate reductase havebeen elucidated with mitochondria from the parasitic nematode, Ascarissuum (Figure 1). In view of the functional significance of PEPCK in theenergy metabolism of parasitic helminths, any change in PEPCK activityfollowing exposure to an anthelmintic drug is important in explaining themode of action of the anthelmintic agent [11].

Protein structure prediction refers to the effort of generating three-dimen-sional models from amino acid sequences using computer algorithms [12, 13].The present work dealing with in silico analysis of homology modeling and3D structure prediction of PEPCK in Ascaris suum will provide insight intoits structure and aid in drug designing.

MATERIALS AND METHODSThe following important work utilizes the protocol involved in in silico 3Dmodeling of Phosphoenolpyruvate carboxykinase (PEPCK) enzyme.

Modeller 9v10The most widely used software for homology (theoretical) modeling; also

Sequence Retrieval and Selection of templateThe amino acid sequence of the target protein was obtained from NCBIdatabase (http://www.ncbi.nlm.nih.gov/protein) with accession numberAAA29378.1. This protein was reported to have 652 amino acid residues.The template selection was performed using BLASTp with PDB database(http://www.rcsb.org/pdb/home/home.do). The protein sequence with a 95%query coverage and E-value of 0 was selected as template. The templatewas of rat cytosolic pepck in complex with beta-2 sulfopyruvate and GTPwhich has a 3D structure with PDB file name 3DT7.

Modeling of 3-D structureThe 3D structures of query sequence were generated by the process ofHomology Modeling using the templates, 3DT7. Five PDB structures weregenerated by modeler. These structures were submitted to Rampage Serverand best protein was selected based on low percentage in outlier region.First we aligned the query sequence with the sequence of the templateprotein Chain-A using the ALIGN2D command of Modeller 9v10 and gen-erated five models. The models generated were accessed for geometricalerrors, energy assessment and Ramachandran Plot analysis and the best

Rampage A trusted server for Ramachandran Plot analysis only to identify the bestprotein generated by homology modeling [16].

3d2GO Server3d2GO server was used for prediction of functions of the predicted modelusing sequence and structure in the reference of Gene Ontology. It predictsthe function of the protein using sources of information like overall topo-logical similarity to structures with known function, geometric and residuesimilarity of predicted functional sites to regions of known structures andsequence homology to functionally annotated sequences [17].

has option to take three templates as input for model generation [14, 15]

(http://salilab.org/index.html).



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Figure 1. Showing potential role of target enzymes phosphoenolpyruvate carboxykinase in parasite. NADH-fumarate reductase system inAscaris suum adult mitochondria. In the phosphoenolpyruvate carboxykinase (PEPCK)- succinate pathway, phosphoenolpyruvate (PEP) pro-duced by a glycolytic process is carboxylated to form oxaloacetate and is then reduced to malate. The cytosolic malate is transported into themitochondria, where it is first reduced to fumarate, and finally to succinate by the rhodoquinol-fumarate reductase activity of complex II. Theterminal step is catalyzed by the NADH-fumarate reductase system comprising complex I, rhodoquinone (RQ), and complex II. Aerobic andanaerobic respiratory chains are boxed in dotted lines.

Figure 2. Cartoon representation of superimposition of homology model(query.pdb) in white colour and template, 3DT7.pdb in pink colourusing chimera.

Figure 3. QMEAN score is a composite score consisting of a linearcombination of 6 terms. The pseudoenergies of the contributing termsare given above together with their Z-scores with respect to scoresobtained for high-resolution experimental structures of similar sizesolved by X-ray crystallography. The Red Cross is the target modelstructure whose Z score is 0.65.



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Figure 4. Prediction of secondary structure of model enzyme phosphoenolpyruvate carboxykinase using Swiss Protein3D assessment server.

model among the five was further refined using loop refinement commandin Modeller 9 v10. Generated models were also evaluated by online Swissmodel server and protein 3D evaluation tool [18, 19, 20].

Evaluation of structureAfter building the protein 3D structure, in order to assess the overall stereochemical quality of the modeled protein, side chain analysis was performedusing the program PROCHECK [21, 22, 23]. ERRAT is a protein structureverification algorithm that is especially well-suited for evaluating the progressof crystallographic model building and refinement [24]. The models gener-ated were validated for geometrical errors, energy assessment using theSwiss Model Assessment Server and ANOLEA. At the end, we perform acomparative analysis of the models generated between the template struc-ture, initial model generated and the refined model and their structure werealigned and study their structural difference of the backbone.

Binding Sites IdentificationSites of binding in proteins usually lie in cavities or on the polar surface.The size and shape of protein surface dictates the three-dimensional geom-etry of ligand that must binds due to intra atomic forces between them. Thebinding of a ligand typically serves as a mechanism for chemical modifica-tion or conformational change of protein.

Secondary structure predictionAfter building the protein 3D structure, the secondary structure predictionwas carried out using Swiss 3 D protein assessment server, a highly accu-rate method for protein secondary structure prediction.

ProQ - Protein Quality PredictorProQ is a neural network based predictor that based on a number of struc-tural features predicts the quality of a protein model. ProQ is optimized tofind correct models in contrast to other methods which are optimized tofind native structures. Two quality measures are predicited LGscore and MaxSub.

RESULTSAfter homology modeling the obtained total five PDB protein files wereanalyzed with Rampage Ramachandran Plot server. The results obtainedwere as follows:Model 1:Number of residues in favoured region (~97.0% expected): (93.1%)Number of residues in allowed region (~2.0% expected): (4%)Number of residues in outlier region: (2.1%)Model 2:Number of residues in favoured region (~98.0% expected): (96.0%)Number of residues in allowed region (~2.0% expected): (3%)Number of residues in outlier region: (1.6%)



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Main- Chain Parameters

Figure 5. Main chain parameters of modeled Phosphoenolpyruvate carboxykinase enzyme.



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Figure 6. Active site prediction of Phosphoenolpyruvate carboxykinaseenzyme using Molegro Virtual Docker 2010.4.0.0 software (Trial ver-sion).

Figure 7. Showing potential biological activity of model Phospho-enolpyruvate carboxykinase enzyme predicted by 3d2GO server.

Figure 8. Showing of final 3D model structure of the Phosphoenolpyru-vate carboxykinase (PEPCK).

Model 3:Number of residues in favoured region (~98.0% expected): (97.6%)Number of residues in allowed region (~2.0% expected): (1.85%)Number of residues in outlier region: (1.1%)Model 4:Number of residues in favoured region (~98.0% expected): (94.3%)Number of residues in allowed region (~2.0% expected): (4 %)Number of residues in outlier region: (1.8%)Model 5:Number of residues in favoured region (~98.0% expected): (96.2%)Number of residues in allowed region (~2.0% expected): (4.3%)Number of residues in outlier region: (2.7%)

Model 3 was selected as the best model as per Ramachandran Plot analysissince it has least number of residues in outlier region. From the above data

it was clear that the homology model done by Modeler of model number 3was the perfect among the others, so we proceed for In Silico active site andfunction prediction.

A superimposed image (Figure 2) of the query and template sequence wasalso carried out to find its resemblance using chimera software. The twostructures seemed to be very much similar to each other. The superimposedstructure clearly showed to build from the same template. This homologymodel number 3 represents 467 numbers of hydrogen bonds, Free energyof folding -632.68 Kcal/mol, Helix 165( 26%), Beta sheet 193 ( 31%), Coil258 ( 41%) and Turn 160( 25%) predicted by VADAR server [25]. The datagenerated by VADAR statistical analysis and protein features are shown inTable 1 and 2. Following superimposition, the sequence was comparedwith non-redundant set of PDB structures, where the z-score was recorded.The graph was plotted between Q Mean score and the size of the nucle-otide (Figure 3). Results of z-scores and QMEAN (Table 3) specify thereliability of the model. The secondary structure prediction was carried outusing Swiss 3D protein assessment server and shown in the Figure 4. TheStereo-chemical properties checks are performed by PROCHECK whichreveals the most significant data shown in Figure 5. The overall qualityfactor of modeled protein was done by ProQ software which showed ex-tremely good structure based on predicted LG score 6.556 and Maxsub0.563.

The cavities highlight the key residues in the protein, mainly with respectto its functional site. The cavities prediction data showed the presence ofone large cavity (Figure 6) with volume 322.56 Å3 and surface areas 988.86Å2. The amino acid present in the cavity at the Proximity of 2.74 Å are His541, Phe 535, Lys 492, Trp 534, Arg455, Phe543, Thr484, Thr490, Asn551,Gly 547, Leu 311, Gly 307, Lys 308, Leu311, Ala305, Ala485, Tyr548,Asn310, Thr 309, Val535, Pro 421, Gly 352, Arg 424, Gly 356, Pro355,Lys417, Glu389 and Pro 546. Function prediction of best model structureusing 3d2GO server was carried out which is shown in the Figure 7. Thegenerated model (Figure 8) was submitted in the PMDB Protein ModelDatabase (http://www.caspur.it/PMDB) bearing Model ID PM0077785.ERRAT structural quality factor and Ramachandran plot is shown in Fig-ure 9 and 10.



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Figure 10. Ramachandran plot of the Psi/Phi distribution of the Phosphoenolpyruvate carboxykinase (PEPCK). The favored and most favoredregions are yellow and red, respectively. Green is the generally allowed region and disallowed region is white.

Figure 9. Showing ERRAT overall structural quality factor.



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DISCUSSIONMost attention has been paid to the pathways of respiratory metabolism inparasites, since maintenance of high energy levels is essential to an organism’ssurvival. Respiratory metabolism is thus a prime target for anthelminticattack. The PEP-succinate pathway is considered the major source of ATPanaerobically in parasitic helminths [26, 27]. Any anthelmintic drug whichcould cause adverse changes in the respiratory metabolism of a parasite butnot in the host is said to be a potential anthelmintic drug. A variety ofanthelmintic drugs have been shown to inhibit some enzymes of thesepathways, which mainly include phosphoenolpyruvate carboxykinase thistarget is a particularly important one since its properties differ signifi-cantly from those of the host. Therefore in the present study it is plannedto model the 3D structure of PEPCK which 3D structure is not available.Homology modeling is currently the most accurate computational methodto predict protein structure. This system constructs a structural model fora target protein sequence from a template structure of a homologous pro-tein [28]. For this method to work, the target and template structures musthave a sequence identity over 30%.

The objective of enzyme inhibition needs structural characterization fromits 3D structure in rational structure based drug design. Homology model-ing produced high quality structural models when the target and templatesare closely related. Enzyme structure modeling undertaken in present workcould be a tool to study better structural characteristics of PEPCK for drugdesign community. Phosphoenolpyruvate carboxykinase is an enzyme inthe lyase family (EC 4.1.1.32). In the present study the PEPCK enzyme ofAscaris suum is modeled with the rat cytosolic PEPCK in complex withbeta-2 sulfopyruvate and GTP which has a 3D structure with PDB file

Table 1. Data showed the VADAR statistics of refined model proteinPhosphoenolpyruvate carboxykinase (PEPCK).

Statistic Observed Expected

Residue in phi psi core 560 (90%) 554(90%)Residue in phi psi allowed 55 (8%) 43(7%)Residue in phi psi generous 1 (0%) 6(1%)Residue in phi psi outside 0 (0%) 0(0%)Residue in omega core 570 (92%) 591(96%)Residue in omega allowed 38 (6%) 18(3%)Residue in omega generous 5 (0%) 0(0%)Residue in omega outside 2 (0%) 6(1%)Packing defects 47 43Free energy of folding -602.68 -593.82Residue 95% buried 221 275Buried charges 17 0

Table 2. The structural information of the model protein Phospho-enolpyruvate carboxykinase (PEPCK).

Alpha helix : 113 is 17.57% 310 helix : 0 is 0.00% Pi helix : 0 is 0.00% Beta bridge : 0 is 0.00% Extended strand : 186 is 28.93% Beta turn : 0 is 0.00% Bend region : 0 is 0.00% Random coil : 344 is 53.50% Ambigous states : 0 is 0.00% Other states : 0 is 0.00%

Table 3. Depiction of total QMEAN value and Z- score.

Scoring function term Raw score Z-score

C-beta interaction energy -116.57 -1.11All-atom pairwise energy -13330.44 -0.93Solvation energy -67.24 0.63Torsion angle energy -173.42 0.06Secondary structure agreement 80.0% 0.25Solvent accessibility agreement 86.0% 0.89Total QMEAN6 score 0.822 0.65

name 3DT7. The modeled enzyme 3-D structure was shown in Figure 8with ribbon view. Modeling studies manifested good stereo chemical place-ment of main chain parameters. Bond angles and bond lengths are underconfined limits (Table 1). Thus, a present study of modeling of Phospho-enolpyruvate carboxykinase protein has brought future prospective to fighthelminthes infection and provide better health standards for community.

The results of the PROCHECK analysis indicate a relatively low percent-age of residues have phi/psi angles in the disallowed ranges (0.8%), thequality of Ramachandran plots is acceptable (Figure 10). The percentage ofresidues in the “core” region of modeled was found to be 98%. The stereochemical quality of the model was found to be satisfactory (Table 3).

Errat analyzes the statistics of non H-bonded interactions between differ-ent atom types and plots the value of the error function versus position ofa residue (Figure 9). The quality of structure was also further assessed byusing ERRAT. ERRAT is a protein structure verification algorithm that isespecially well-suited for evaluating the progress of crystallographic modelbuilding and refinement. Errat is showing an overall quality factor of 97.4.

ACKNOWLEDGEMENTSWe acknowledge the University Grant Commission, New Delhi (India) forproviding Research fellowship in science for meritorious students (F-4-1/2006(BSR)/5-120/2007(BSR), Dated 20th January’09. The author is alsothankful to Tezpur University for organizing and providing complete train-ing for using Modeller v9.10 software.

CONCLUSIONCurrently there is no information available on 3D characterization of PEPCKenzyme which play most important role in parasite energy metabolism.Unavailability of 3-dimensional structure of enzymes is one of the majorhindrances in elucidating the interactions of enzymes with possible inhibi-tors. Comparative homology modeling is promulgated as the most un-swerving computer-based technique for deciphering the 3D structures inthe absence of crystal structure of the protein. In this study the (PEPCK)has been selected for which three dimensional structures is not available atthe protein data bank (PDB). This article describes the in silico analysis ofhomology modeling and 3D structure prediction of PEPCK in Ascarissuum which will provide insight into its structure and aid in drug designing.

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Source of support: Nil, Conflict of interest: None Declared

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