International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 3(6), 29-40, June (2014) Int. Res. J. Biological Sci. International Science Congress Association 29 Computational Approach to Explore Plasmodium falciparum 3D7 Seryl tRNA Synthetase StructureKasturi K., Mallika D.S., Jeevan Amos S., Pavithra V. and Sambasiva Rao K.R.S.Department of Biotechnology, Acharya Nagarjuna University, Guntur, AP, INDIAAvailable online at: www.isca.in, www.isca.me Received 8th November 2013, revised 24th January 2014, accepted 10th February 2014Abstract Malaria remains the leading cause of deaths attributable to a communicable disease globally. The reemergence of drug-resistant to Plasmodium falciparum, which is the most fatal human cerebral malarial parasite, has focused attention on aminoacylation in protein translation. Seryl tRNA Synthetase is the enzyme involved in acylation of Serine amino acid to tRNA. The protein sequence of Seryl tRNA synthetase containing 539 residues was obtained from UniprotKB. The enzyme was characterized using computational tools. The secondary and tertiary (3D) structure of the enzyme was predicted using Seryl tRNA Synthetase from other species and validated by various structural quality validation tools. Results of the current study will provide a deep insight about the structure and function of Seryl tRNA synthetase and aid in drug docking, rational drug designing. Keywords: P.falciparum, aminoacyl tRNase, Seryl tRNA synthetase, 3D structure modelling, cerebral malaria. IntroductionMalaria is the significant health problem affecting millions of people and thousands of lives are at risk every year. It is caused by five species of protozoan parasites of the genus Plasmodiumthat affect humans (P. falciparum, P. ovale, P. vivax, P. malariae and P. knowlesi). The data from the world malaria report 2011 summarizes that among 106 malaria-endemic countries from 6 regions, P. falciparum is the major malarial pathogen causing deathsThe life cycle of the Plasmodium is complex with two major phases: liver and blood. As the infected mosquito bites a human host, the Plasmodium sporozoites migrate to the liver and infect hepatocytes. After replicating within the hepatocyte, the parasites rupture the cell to release merozoites, a stage specialized to infect erythrocytes. However, two subspecies (P. vivax and P. ovale) can assume a dormant state (hypnozoites) within the liver that commonly lasts for months and can last for years. After exiting the liver, the parasite establishes a recurring life cycle in the erythrocytes. During the blood stage, some parasites will differentiate into sexual forms that are transmitted to the next human host by the vector. Each developmental stage is characterized by distinct physiology, and each has varying sensitivity to most drugs3,4. To date stage specific antimalarial chemotherapy is applied for the control and treatment5,6. But the emergence of multi drug resistant strains of Plasmodium towards commercial drugs has made the treatment of disease more critical. The development of resistance occurs in two phases i.e., the development of resistant mutant and the second is multiplication of the resistant parasite resulting in the parasite population that is no longer suitable for treatment. Thus the situations have resulted in the identification of novel drug targets with novel mechanism within the parasite. Basing on the concepts of Flux balance analysis (FBA) to assess the essentiality of genes for an organism7-9 and concepts of choke-points and load points for estimating the reactions that are essential for the organism10,11P. falciparum seryl tRNA synthetase is being considered as a putative target. P. falciparum seryl tRNA synthetase (PF07_0073) belongs to class II aminoacyl-tRNA’s synthetase. It catalyses the reaction between amino acid L-serine and tRNA to form L-seryl-tRNA, which is necessary for the synthesis of various proteins. Inhibition of this enzyme is likely to affect the aminoacyl tRNA biosynthesis pathway of the parasite. Unavailability of the 3 - dimensional structure of enzymes is one of the major hindrances in elucidating the interactions of enzymes with possible inhibitors. Comparative homology modeling is promulgated as the most unswerving computer-based technique for deciphering the 3D structures in the absence of the crystal structure of the protein. This article describes the modeling and validation of seryl tRNA synthetase of P. falciparum which will provide insight into its structure and aid in drug designing. Methodology Selection of Malarial Parasite: P. falciparum 3D7 was selected as the candidate organism for the present study because of 4 reasons. i. Among the five species of Plasmodium, P.falciparum was reported as highest mortality in Africa as well as in South- East Asian regions according to WHO malaria report 2011 (table 1, figure 1). ii. It causes cerebral malaria which is a dreadful disease. iii. Complete genome sequence of P. falciparum is available. iv. P. falciparum showing high frequency of resistance to available drugs in the market. Resistance towards the presently available drugs was due to International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(6), 29-40, June (2014) Int. Res. J. Biological Sci. International Science Congress Association 30 improper identification of P. falciparum and abnormal drug dosage without specificity to the P. falciparum and finally parasite becomes resistant to the available drugs very rapidly. So there is a need for the emergence of new drugs with new targets. This is the main motive of our present study to choose P. falciparum as a target sps. Sequence analysis of Seryl tRNA synthetase of Plasmodiumfalciparum 3D7: Seryl tRNA synthetase protein sequence (Q8IBS3) was collected from UniprotKB, FASTA formatted sequence was retrieved (figure-2) and submitted to pBLAST for similar structural protein against the PDB-RCSB database keeping default parameters like E-value threshold 10, word size 3 and Blosum 62 Matrix. Since the BLAST algorithm detects local as well as global alignments, regions of similarity embedded in otherwise unrelated proteins can be detected12,13. The BLAST result page contains template sequences which were showing structural homology to query sequence (table 2). Among them Seryl-tRNA Synthetase (PDB id: 3QNE_a) from Candida albicans was taken as a template for the present study because it is having an E-value (2e-114) and query coverage (99%), Max identity (38%) were satisfactory. The template protein sequence was derived from uniprotKB for further analysis. Multiple sequence alignment was performed using CLUSTAL_X214.15. Regions of conservation and variation were detected from CLUSTAL_X2 result (figure 3). Functional Characterization of Seryl tRNA synthetase of Plasmodiumfalciparum 3D7: Functional characterization of SeryltRNA synthetase protein sequence was done by finding motif using Eukaryotic Linear Motif and domain analysis was carried out using PROTSCAN. ELMs are very short motifs nothing but regular expressions of a protein, many of them will overpredict, implying that most matches shown are more likely to be false positives than true matches16. To improve the predictive value of ELM, logical filters (or rules) are used based on context information to discriminate between likely true and false positives. PROSCAN is a tool to scan a sequence against the PROSITE database. That is to say, to search for biologically relevant sites and signatures. By its algorithm (shared with PATTINPROT), PROSCAN allows searching with errors. Errors are set by number of mismatches allowed or by a level of significant biological information. This tool is available in NPS (http://npsa-devel.ibcp.fr/NPSA/npsa_proscan.html) 17. All results were tabulated in table 3and table 4,figure 4. Physico-Chemical Characterization of Seryl tRNA synthetase of Plasmodiumfalciparum 3D7: The basic physico-chemical properties of the seryl tRNA synthetase protein sequence were calculated using the ProtParam tool18. Molecular weight, theoretical pI, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, aliphatic index and grand average of hydropathicity19 (GRAVY) was computed by ProtParam. Results were tabulated in table 5, 6 and 7. Secondary Structure prediction of P. falciparum 3D7 Seryl tRNA Synthetase: GOR-IV tool was used to obtain the secondary structure of seryl tRNA synthetase protein http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl? page= npsa_gor4.html)20. Secondary structure prediction is the definition of each residue into either alpha helix, beta sheet or random coil secondary structures. These results were tabulated in table 8 and secondary structural details were shown in figure 5. 3D structure prediction of P. falciparum 3D7 Seryl tRNA Synthetase: 3D structure prediction of P. falciparum 3D7 seryl tRNA synthetase protein was performed using Modeller 9.10(http://salilab.org/modeller/)21, it generates model based on comparative or homology method using user provided alignment of a sequence to be modeled with known related structures. MODELLER automatically calculates a model containing all non-hydrogen atoms and generates a model by satisfaction of spatial restraints22,23,24 and other web based servers such as Geno3D, ESYPred 3D, 3DJIGSAW, SWISS MODEL and CPHmodel were also used to analyze the generated model quality (table 9). Geno 3D: It predicts the 3D structure of an amino acid sequence, an automated protein modeling Web server is used to generate protein 3D model. Gene3D is supplementary to the CATH database, contains proteins from complete genomes which have been clustered into protein families and annotated with CATH domains, Pfam domains and functional information from KEGG, GO, COG, Affymetrix and STRINGS25. ESyPred3D: ESyPred3D is a new automated homology modeling program. The method gets benefit of the increased alignment performances of a new alignment strategy using neural networks. Alignments are obtained by combining, weighting and screening the results of several multiple alignment programs. The final 3D structure is built using the modeling package MODELLER26. 3D-JIGSAW: It is an automated system to build three-dimensional models for proteins based on homologues of known structure from databases (PFAM+PDB+nr) and splits the query sequence into domains. From the template hits, the best covered domain is used to model the query sequence; maximum of 2 models can be generated by this method27-29. SWISS MODEL: It is a fully automated protein structure homology modeling server accessible via the expasy web server. The purpose of this server is to make protein modeling accessible to all biochemist and molecular biologist's world wide30,31,32. International Research Journal of Bi ological Sciences ________________________________________________ Vol. 3(6), 29-40, June (2014) International Science Congress Association CPH Model: CPHmodels-3.0 is a web- server predicting protein 3D- structure by use of single template homology modelling is a group of high performing 3D- prediction tools. Beside its accuracy, one of the important features of the method is its speed. For most queries, the response t ime of the server is less than 20 minutes. Evaluation of the obtained models of Seryl tRNA Synthetase protein: The result was evaluated using different web based validation servers such as PROCHECK and ProSA. PROCHECK: This program assesses the “stereo quality” of a given protein structure. The aim of PROCHECK is to assess how normal or how unusual, the arrangement of the residue geometry in a given protein structure is, as compared with stereo-chemical paramet ers derived from well high-resolution structures34,35 . Results from other web based servers were compared. ProSA: which is frequently employed in protein structure validation, it requires the atomic coordinates (PDB file format) of the model to be evaluated. It uses only the C the input structure, hence it can also be applied to low resolution structures and approximate models obtained early in the structure determination process36. Results and Discussion Selection of Malarial Parasite: P. falciparum target parasite because of its existence. Most of the WHO Graphical representation of statistical data of existence of Malarial parasite 20406080100120 Percentage of occurence ological Sciences ________________________________________________ International Science Congress Association server predicting protein structure by use of single template homology modelling 33. It prediction tools. Beside its accuracy, one of the important features of the method is its ime of the server is less Evaluation of the obtained models of Seryl tRNA Synthetase The result was evaluated using different web based validation servers such as PROCHECK and ProSA. This program assesses the “stereo -chemical quality” of a given protein structure. The aim of PROCHECK is to assess how normal or how unusual, the arrangement of the residue geometry in a given protein structure is, as compared ers derived from well -refined, . Results from other web based which is frequently employed in protein structure requires the atomic coordinates (PDB file format) uses only the C -alpha atoms of the input structure, hence it can also be applied to low resolution structures and approximate models obtained early in the P. falciparum was selected as target parasite because of its existence. Most of the WHO regions had high intensity of P. falciparum reported. Statistical data of this was tabulated in table 1, graphical representation was given in divides total 101 malaria affected countries in 6 regions, among these regions highest rates of P. falciparum reported from the African region and least was reported in the European region. Table- 1 Percentage o f existence of Malarial Parasite according to WHO Malaria report Percentage of Existence of Malarial Parasite Regions P. faiciparum South east Asia African 96.795 Eastern Mediterranean Western Pacific European 0.166 American 21.428 Selection of Target Protein and Retrieval: synthetase is an enzyme involved in acylation of serine to trna in protein translation. This is a putative drug target to design lead molecules for the inhibition of growth of The protein sequence of target protein was retrieved from Uniprotkb as given in figure 2. Figure-1 Graphical representation of statistical data of existence of Malarial parasite RegionsExistance of Malarial Parasite Plasmodium faiciparum Others ological Sciences ________________________________________________ ISSN 2278-3202 Int. Res. J. Biological Sci. 31 P. falciparum infections were reported. Statistical data of this was tabulated in table 1, graphical representation was given in figure 1. Generally WHO divides total 101 malaria affected countries in 6 regions, among P. falciparum infections were reported from the African region and least was reported in the 1 f existence of Malarial Parasite according to WHO Malaria report - 2011 Percentage of Existence of Malarial Parasite P. faiciparum Others 44.9 56.1 96.795 3.315 59.1 41.9 54.5 45.5 0.166 99.944 21.428 79.682 Selection of Target Protein and Retrieval: Seryl tRNA synthetase is an enzyme involved in acylation of serine to trna in protein translation. This is a putative drug target to design lead molecules for the inhibition of growth of P. falciparum. The protein sequence of target protein was retrieved from Graphical representation of statistical data of existence of Malarial parasite Plasmodium faiciparum International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(6), 29-40, June (2014) Int. Res. J. Biological Sci. International Science Congress Association 32 Sequence analysis of Seryl tRNA synthetase of Plasmodiumfalciparum 3D7: Blast Results: Upon performing the pBLAST total top 100 hits were listed in the result page as descending order of similarity score. Among them first 10 with the PDB ID of BLAST hits, similarity score, query coverage and e values were tabulated in table 2. The first and best similarity structure is 3VBB which is a human protein not taken as template because the generated model was for human pathogen if human protein is taken as a template then there was chance to inhibit the human protein also when the lead molecule used for target protein. So by considering this even though it shows highest similarity, it was not taken as a template to generate the model. Clustal X2 Results: From the BLAST results 3QNE was taken as a template for the target protein to generate structure model by comparative modeling. This template had queried coverage, Similarity score and e-values 99%, 351 and 2e-114 respectively were quite satisfactory. So the 3QNE protein sequence was retrieved and subjected to Multiple Sequence Alignment with the target or query protein using the Clustal X2 tool. The result of Clustal X2 was reported in figure 3. Figure-2 Sequence of Target protein: Seryl tRNA Synthetase Table-2 pBlast result- First 10 hits of the query proteinPDB ID Query Coverage (%)Similarity Score E-Value Max Identity (%) 3VBB99 381 3e-125 39 3QNE99 351 2e-114 38 3QQ599 349 1e-113 38 3LSQ99 338 4e-109 36 2DQ099 277 5e-86 32 2DQ398 234 2e-70 31 1SRY61 164 2e-44 28 3ERR61 166 2e-44 28 1WLE65 133 1e-33 26 3MEY13 31.6 1.3 24 Figure-3 Multiple sequence alignment of Target with 3QNE using Clustal X2 tool �tr|q8ibs3|q8ibs3_plaf7 seryl-trna synthetase, putative os=plasmodium falciparum (isolate 3d7) gn=pf07_0073 pe=3 sv=1 MVLDINLFRKEKGGNPDKIKESERKRYHDENNVDKVIEYDDKWRKCIFELEELKKNINMINKEIGNKKKVDKNADVEDLKKKSLNIKEEIPKYQLKEKELLKERNKYISKIGNLLNIKVVCSDNEDNNKIVKTWGECKILPACEENDNSIHDNVVNSNNIKRETLNNEVDNKKKIKYYYHYDLLRKIGGANFKKGIQVAGHRGYYLTGAGFLLHNAILQYALNFLVNKKYIPVYPPFFMKKNIMEECAELDDFEETLYKIPSTSNSTLSSQQVSTSPTKISSQADIKDDTTCNSQKKTNIPSNEDLTRDDLFLIATSEQPLCALHKDETIESKRLPLKYAGFSSCFRKEAGAHGKDIRGILRVHQFDKVEQFCIALPQHSNKIHEEMIQTCEEFYQSLNIPYRIVSIVSGALNNAASIKYDLEGFFPTSNQYRELVSCSNCTDYQSINLNIRYSDSSIKINDLNKNTNLNDEMDSEYEHFLTNFNTENKYHVHLLNGTMVAAQRFLCCLLENYQNGEGIVVPEKLRPYMNNMDFIPFME International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(6), 29-40, June (2014) Int. Res. J. Biological Sci. International Science Congress Association 33 Functional Characterization of Seryl tRNA synthetase of Plasmodiumfalciparum 3D7: The function of Sery tRNA synthetase protein of P. falciparum 3D7 was analyzed by submitting the amino acid sequence to ELM server and Proscan server. Based on EML and PROSCAN results, the following motif and domains were assigned to target protein amino acid sequence (table 3 and 4, figure 4). Table-3 Motif analysis of P. falciparum 3D7 Seryl tRNA synthetase Element Name Position Element Description Pattern LIG_CYCLIN_1 138-141 [A] 433-436 [A] Substrate recognition site that interacts with cyclin and thereby increases phosphorylation by cyclin/cdk complexes. Predicted protein should have the MOD_CDK site. Also used by cyclin inhibitors [RK].L.{0,1}[FYLIVMP] LIG_eIF4E_1 178-184 [A] Motif binding to the dorsal surface of eIF4E. Y....L[VILMF] LIG_FHA_2 131-137 [A] Phosphothreonine motif binding a subset of FHA domains that have a preference for an acidic amino acid at the pT+3 position. ..(T)..[DE]. LIG_TRAF2_1 142-145 [A] Major TRAF2- binding consensus motif. Members of the tumor necrosis factor receptor (TNFR) superfamily initiate intracellular signaling by recruiting the C-domain of the TNFR-associated factors (TRAFs) through their cytoplasmic tails [PSAT].[QE]E MOD_N-GLC_1 439-444 [A] 495-500 [A] Generic motif for N-glycosylation. Shakin-Eshleman et al. showed that Trp, Asp, and Glu are uncommon before the Ser/Thr position. Efficient glycosylation usually occurs when ~60 residues or more separate the glycosylation acceptor site from the C-terminus .(N)[^P][ST].. MOD_PKA_1 161-167 [A] Main preference for PKA-type AGC kinase phosphorylation. [RK][RK].([ST])[^P].. MOD_PKA_2 161-167 [A] 451-457 [A] Secondary preference for PKA-type AGC kinase phosphorylation. .R.([ST])[^P].. MOD_PLK 146-152 [A] 454-460 [A] Site phosphorylated by the Polo-likekinase .[DE].([ST])[ILFWMVA].. MOD_SUMO 160-163 [A] Motif recognised for modification by SUMO-1 [VILMAFP](K).E TRG_ENDOCYTIC_2 181-184 [A] 432-435 [A] 444-447 [A] 477-480 [A] Tyrosine-based sorting signal responsible for the interaction with mu subunit of AP (Adaptor Protein) complex Y..[LMVIF] - - TRG_NLS_Bipartite_1 161-177 [A] Bipartite variant of the classical basically charged NLS. [KR][KR].{7,15} [^DE]((K[RK])| (RK))(([^DE] [KR])| ([KR][^DE]))[^DE] TRG_NLS_MonoExtC_3 171-176 [A] Monopartite variant of the classical basically charged NLS. C-extended version. [^DE]((K[RK])| (RK))(([^DE] [KR])| ([KR][^DE])) (([PKR])|([^DE] [DE])) TRG_NLS_MonoExtN_4 172-177 [A] Monopartite variant of the classical basically charged NLS. N-extended version. (([PKR]. {0,1}[^DE])| ([PKR]))((K[RK])| (RK))(([^DE] [KR])| ([KR][^DE]))[^DE International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(6), 29-40, June (2014) Int. Res. J. Biological Sci. International Science Congress Association 34 Figure-4 Schematic representation of different types of predicted regions of target proteinTable-4 Domains obtained in P. falciparum 3D7 Seryl tRNA synthetase Protein using PROSCAN Domine name Prosite Accession Number Randomized probability Start position End position Sequence of Prosite Pattern N-Gylcosylation Site PS00001 5.138e-03 255 268 NSTL N-{P}-[ST]-{P} [N is Gylcosylation site] 440 443 NCTD 496 499 NGTM cAMP- and cGMP-dependent protein kinase phosphorylation site PS00004 1.572e-03 80 83 KKKS [RK](2)-x-[ST] [S or T is the phosphorylation site] 161 164 KRET Protein kinase C phosphorylation site PS00005 1.423e-02 22 24 SER [ST]-x-[RK] [S or T is the phosphorylation site] 294 296 SQK 332 334 SKR 380 382 SNK 417 419 SIK 457 459 SIK Casein kinase II phosphorylation site PS00006 1.482e-02 122 125 SDNE [ST]-x(2)-[DE] [S or T is the phosphorylation site] 133 136 TWGE 149 152 SIHD 282 285 SQAD 302 305 SNED 307 310 TRDD 390 393 TCEE 475 478 SEYE Tyrosine kinase phosphorylation site. PS00007 4.074e-04 (min) 20 27 KESERKRY [RK]-x(2)-[DE]-x(3)-Y or [RK]-x(3)-[DE]-x(2)-Y [Y is the phosphorylation site] 4.083e-04 (max) N-myristoylation site PS00008 1.397e-02 112 117 GNLLNI G-{EDRKHPFYW}-x(2)-[STAGCN]-{P} [G is the N-myristoylation site] 195 200 GIQVAG 410 415 GALNNA 497 502 GTMVAA International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(6), 29-40, June (2014) Int. Res. J. Biological Sci. International Science Congress Association 35 Physico chemical characterization of target protein: ProtParam computes various physico-chemical properties of a protein from its sequence. The molecular weight, theoretical pI, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, aliphatic index and grand average of hydropathicity (GRAVY) were computed by ProtParam listed in table 5, 6, 7. Table-5 Amino acid composition of Seryl tRNA synthetase of P. falciparum 3D7Amino acid Number Percentage Ala (A) 203.7% Arg (R) 183.3% Asn (N) 5510.2% Asp (D) 336.1% Cys (C) 142.6% Gln (Q) 173.2% Glu (E) 468.5% Gly (G) 213.9% His (H) 132.4% Ile (I) 438.0% Leu (L) 478.7% Lys (K) 5810.8% Met (M) 101.9% Phe (F) 213.9% Pro (P) 173.2% Ser (S) 346.3% Thr (T) 213.9% Trp (W) 20.4% Tyr (Y) 254.6% Val (V) 244.5% Pyl (O) 00.0% Sec (U) 00.0% Table-6 Atomic composition of P. falciparum 3D7 Seryl tRNA synthetase Element Symbol No. of atoms Carbon C 2762 Hydrogen H 4357 Nitrogen N 751 Oxygen O 850 Sulfur S 24 Secondary structure prediction of target protein: GOR-IV program that was used to predict secondary structures in P. falciparum 3D7 suggest that it contained more helices than beta sheets in target protein (figure 5). Three dimensional structure of target protein of P. falciparum 3D7 was predicted (figure 6) by modeler 9.10 using a template of Seryl tRNA synthetase of Candida albicans (PDB ID: 3QNE keeping default parameters. Other online servers also used to generate the best model. Servers and their templates were listed in table 9. 3vbb is a human protein so in the present study it was not taken as a template. Table-7 Physico-chemical properties of Seryl tRNA synthetase of P. falciparum 3D7Property Value Number of amino acids 539 Molecular weight 62453.9 Theoretical pI 6.53 Total number of negatively charged residues (Asp + Glu) 79 Total number of positively charged residues (Arg + Lys) 76 Total number of atoms 8744 Aliphatic index 81.74 Grand average of hydropathicity (GRAVY) -0.682 Ext. coefficient 49125 Ext. coefficient 48250 Estimated half-life 30 hours Instability index (II) 42.72 Formula 2762 H 4357 N 751 O 850 S 24 Table-8 Percentage and type of Secondary structures present in P. falciparum 3D7 Seryl tRNA Synthetase Secondary structure Stretch Percentage Alpha helix (Hh) 15628.94% 310helix (Gg)0.00% Pi helix (Ii)0.00% Beta bridge (Bb) 00.00% Extended strand (Ee)10519.48% Beta turn (Tt) 00.00% Bend region (Ss) 00.00% Random coil (Cc) 27851.58% Ambiguous states 00.00% Other states 00.00% Table-9 Different servers used to generate best model of the target protein Server Template Modeller 9.10 3QNE Geno3D 3QNE ESYPred3D 3QNE 3DJIGSAW 3ISS Swiss model 3VBB Cph model 3VBB International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(6), 29-40, June (2014) Int. Res. J. Biological Sci. International Science Congress Association 36 Figure-5 Secondary structure prediction of Target protein of P. falciparum 3D7 by GOR-IV tool Figure-6 3D structure of P. falciparum 3D7 Seryl tRNA synthetase International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(6), 29-40, June (2014) Int. Res. J. Biological Sci. International Science Congress Association 37 The model was stereo chemically evaluated using the program PROCHECK. Through the inspection of the Psi/Phi angles of a Ramachandran plot obtained from this analysis (figure 7), the backbone conformation of the model was evaluated. The overall conformation of the backbone was in good agreement with the stereochemistry, which was also found to be reliable (figure 8).Figure-7 Ramachandran plot of modeled protein obtained from Modeller 9.10 using PROCHECK Plot statistics Residues in most favoured regions [A,B,L] 441 88.4% Residues in additional allowed regions [a,b,l,p] 53 10.6% Residues in generously allowed regions [~a,~b,~l,~p] 3 0.6% Residues in disallowed regions 2 0.4% Number of non-glycine and non-proline residues 499 100.0% Number of end-residues (excl. Gly and Pro) 2 Number of glycine residues (shown as triangles) 21 Number of proline residues 17 Total number of residues 539 Based on an analysis of 118 structures of resolution of at least 2.0 Angstroms and R-factor no greater than 20%, a good quality model would be expected to have over 90% in the most favoured regions. International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(6), 29-40, June (2014) Int. Res. J. Biological Sci. International Science Congress Association 38 Figure-8 Overall Quality and Local quality of modeled protein obtained from Modeller 9.10 using ProSA with Z- Score Table-10 Model quality of Target protein obtained from different servers based on Ramachandran Plot using PROCHECK, Z- Score using ProSA Server ProSA PROCHECK Z- Score Residues in most favored regions (%) Residues in additional allowed regions (%) Residues in generously allowed regions (%) Residues in disallowed regions (%) Modeller 9.10 -7.48 88.4 10.6 0.6 0.4 Geno3D -7.48 64.9 25.3 5.8 4.0 ESYPred3D -7.48 77.1 11.6 6.7 4.6 3DJIGSAW -7.48 64.9 25.3 5.8 4.0 Other web based servers were also employed to generate the 3 dimensional structure of P. falciparum 3D7 Seryl tRNa synthetase and the results were compared. Based on these evaluations, we conclude that model obtained using Modeller 9.10 is superior as compared to models generated using other web based servers. Conclusion WHO statistics indicates the severity of malaria, as the pre-eminent tropical disease and it is rated as one of the top three killers among communicable diseases. Emergence of drug resistance strains and the inability of stage specific commercially available anti malarial drugs are one of the greatest coercion to our ability to battle against malaria. The situation continues to be more frightening, with the geographical spread of resistance widening to previously unaffected areas and a ruthless augmentation both in the incidence and degree of drug resistance. Thus there is an urgent need for the development of highly selective and efficacious antimalarial therapies designed against novel Plasmodial targets. The Seryl tRNA synthetase enzyme from P. falciparum represents one such target of interest. A refined model of Seryl tRNA synthetase was generated using the crystal structures of closely related homologues Candida seryl tRNA synthetase. The structural accuracy of the model was extensively validated using protein structure checking tools. This hypothesis describes the modeling and validation of seryl tRNA synthetase of P. falciparum, which will provide insight into its structure and aid in drug designing. Acknowledgement The authors thank the Department of Biotechnology, Govt. of India for the financial assistance provided to carry out the project work. The authors would also like to thank Acharya Nagarjuna University, Guntur, India for providing the congenial International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(6), 29-40, June (2014) Int. Res. J. Biological Sci. International Science Congress Association 39 environment and also for extending the lab facilities to carry out the work. References1.World Malaria Report 2011 (Geneva: World Health Organization) WHO, (2011)2.White N.J., Determinants of relapse periodicity in Plasmodium vivax malaria, J. Malaria., (10), 297 (2011)3.Wells T.N., Alonso P.L. and Gutteridge W.E., New medicines to improve control and contribute to the eradication of malaria, Nat. Rev. Drug Discov., (8), 879–891 (2009)4.Bhatt T.K., Structural Characterization of Histone Deacetylase from Plasmodium falciparum, ISCA J. Biological Sci, (1), 65-68 (2012) 5.Laurence Florens et al., A proteomic view of the Plasmodium falciparum life cycle, Nature, (419), 520–526 (2002) 6.Cecile Crosnier, et al., Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum,Nature.,(480), 534–538 (2011)7.Becker, S.A., Feist, A.M., Mo, M.L., Hannum, G., Palsson, B.O. and Herrgard, M.J., ‘Quantitative prediction of cellular metabolism with constraint-based models: The COBRA Toolbox’, Nat. Protoc., (2),727–738(2007) 8.Edwards J.S. and Palsson B.O., Metabolic flux balance analysis and the in silicoanalysis of Escherichia coli K-12 gene deletions, BMC Bioinf. (1:1), (2000) 9.Kauffman K.J., Prakash P. and Edwards J.S., Advances in flux balance analysis, Curr. Opin. Biotechnol., (14), 491–496 (2003)10.Puntervoll P., Linding R., Gemünd C., Chabanis-Davidson S., Mattingsdal M., Cameron S., Martin D. M. A., Rahman S.A. and Schomburg D.‚ Observing local and global properties of metabolic pathway - load points and choke points in the metabolic networks, Bioinformatics,10(22), 1767–1774 (2006)11.Yeh I., Hanekamp T., Tsoka S., Karp P.D. and Altman R.B., Computational analysis of Plasmodium falciparummetabolism: organizing genomic information to facilitate drug discovery, Genome Res., (14), 917–924 (2004) 12.Altschul S.F., Madden T.L., Schäffer A.A., Zhang J., Zhang Z., Miller W. and Lipman D.J., Gapped BLAST and PSIBLAST: a new generation of protein database search programs, Nucleic Acids Res., (25), 3389-3402 (1997)13.Katrina C. Martinez, Franco G. Teves and Ma. Reina Suzette B. Madamba, Sequence Analysis of Putative luxS Gene Involved in Prodigiosin Biosynthesis from Philippine Local Strains of Serratia marcescens, ISCA J. Biological Sci, (2), 13-19 (2013) 14.Thompson J.D., Gibson T.J., Plewniak F., Jeanmougin F. and Higgins D.G., The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools, Nucleic Acids Research, (25), 4876-4882 (1997) 15.Maithri S.K., Ramesh K.V., Dieudonné Mutangana and Deshmukh Sudha., Molecular Modeling and Docking Studies of PirB Fusion Protein from Photorhabdus Luminescens, ISCA J. Biological Sci, (1), 7-18 (2012) 16.Ausiello G., Brannetti B., Costantini A., Ferrè F., Maselli V., Via A., Cesareni G., Diella F., Superti-Furga G., Wyrwicz L., Ramu C., McGuigan C., Gudavalli R., Letunic, I., Bork, P., Rychlewski, L., Küster, B., Helmer-Citterich, M., Hunter, W. N., Aasland, R. and Gibson, T. J., ELM server: a new resource for investigating short functional sites in modular eukaryotic proteins, Nucleic Acids Res (31), 3625-3630 (2003) 17.Combet C., Blanchet C., Geourjon C. and Deléage G.NPS@: network protein sequence analysis. Trends Biochem Sci.,(25:3),147-150 (2000)18.Gasteiger E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M.R., Appel, R.D.and Bairoch, A., ‘Protein Identification and Analysis Tools on the ExPASy Server’, The Proteomics Protocols Handbook, 18,571-607 (2005)19.Kyte J. and Doolittle R.F., A simple method for displaying the hydropathic character of a protein, J. Mol. Biol, (157), 105-132 (1982) 20.Garnier J., Osguthorpe DJ. and Robson B., Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins, J Mol Biol, (120), 97-120 (1978)21.Marti-Renom, M.A., Stuart, A., Fiser, A., Sanchez, R., Melo, F. and Sali, A., Comparative protein structure modeling of genes and genomes, Annu. Rev. Biophys. Biomol. Struct, 21(29),291-325(2000)22.Sali A. and Blundell T.L., Comparative protein modelling by satisfaction of spatial restraints, J. Mol. Biol.,(234), 779-815(1993) 23.Fiser, A., Do, R.K. and Sali, A., Modeling of loops in protein structures, Protein Science, (9),1753-1773(2000) 24.Bhatt T.K., Structural Studies on Mitogen Activated Protein Kinase from Plasmodium falciparum, ISCA J. Biological Sci, (1), 42-46 (2012) 25.Combet C., Jambon M., Deleage G. and Geourjon, C.‘Geno3D: automatic comparative molecular modelling of protein, Bioinformatics, (18), 213-214 (2002)26.Lambert, C., Leonard, N., De Bolle, X.and Depiereux, E., ‘ESyPred3D: Prediction of proteins 3D structures’, Bioinformatics, (18:9), 1250-1256 (2002) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(6), 29-40, June (2014) Int. Res. J. Biological Sci. International Science Congress Association 40 27.Bates, P.A., Kelley, L.A., MacCallum, R.M. and Sternberg, M.J.E.,‘Enhancement of Protein Modelling by Human Intervention in Applying the Automatic Programs 3D-JIGSAW and3D-PSSM’, Proteins: Structure, Function and Genetics, (5),39-46(2001)28.Bates, P.A. and Sternberg, M.J.E.,‘Model Building by Comparison at CASP3: Using Expert Knowledge and Computer Automation’, Proteins: Structure, Function and Genetics, (3),47-54(1999). 29.Contreras-Moreira, B., Bates, P.A., ‘Domain Fishing: a first step in protein comparative modelling’, Bioinformatics, (18),1141-1142(2002)30.Arnold, K., Bordoli, L., Kopp, J. and Schwede, T.‘The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling”, Bioinformatics, (22),195-201(2006). 31.Schwede, T., Kopp, J., Guex, N. and Peitsch, MC., ‘SWISS-MODEL: an automated protein homology-modeling server’, Nucleic Acids Research., (31),3381-3385(2003) 32.Guex, N. and Peitsch, M. C., ‘SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling’., Electrophoresis., (18),2714-2723(1997)33.Nielsen, M., Lundegaard, C., Lund, O. and Petersen, TN., ‘Remote homology modeling using structure guided sequence profiles’, Nucleic Acids Research.,33(38),576-81(2010) 34.Laskowski, R A., MacArthur, M W., Moss, D S. and Thornton, J M. (1993), ‘PROCHECK - a program to check the stereochemical quality of protein structures’., J. App. Cryst., (26),283-291(1993)35.Laskowski, R A., Rullmannn, J A., MacArthur, M W., Kaptein, R. and Thornton, J M., ‘AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR’, J Biomol NMR, 35(8),477-486(1996) 36.Sippl, M.J., Recognition of Errors in Three-Dimensional Structures of Proteins, Proteins (17),355-362(1993)