Research Journal of Recent Sciences _________________________________________________ ISSN 2277-2502 Vol. 2(2), 40-49, February (2013) Res. J. Recent Sci. International Science Congress Association 40 Theoretical structure prediction of TcaA from Photorhabdus luminescens and aminopeptidase N receptor from Helicoverpa armigeraMaithri S.K., Ramesh K.V. and Mutangana Dieudonné Department of Biotechnology, Center for Postgraduate studies, Jain University, Jayanagar, Bangalore – 560011, INDIAAvailable online at: www.isca.in Received 12th October 2012, revised 22nd November 2012, accepted 22nd December 2012Abstract Due to resistance developed by various agricultural pests towards Bacillus thuringiensis (BT) toxins, there is a necessity of developing alternative biopesticide. The TcaA toxin produced by Photorhabdus luminescens is a high molecular weight insecticidal toxin having toxicity against wide range of agricultural pests. Phylogenetic tree constructed for TcaA revealed that this toxin does not have any ancestral relationship with BT toxins. Present study focused on the modeling the TcaA toxin from Photorhabdus luminescens and aminopeptidase N receptor from Helicoverpa armigera using various computational approaches. Structural validation using various tools such as ProSA and PROCHECK revealed that and angles of these theoretical models were present in the core and allowed region. The theoretical toxin structure was subsequently docked onto the homology modeled aminopeptidase N receptor. Outcome of the docking study showed that first domain of TcaA had highest docking energy when compared to remaining domains. Keywords: TcaA, APN, docking, molecular modelling, domain. Introduction Various problems are associated with the application of synthetic insecticides. Many of these insecticides are non-biodegradable, undergo slow degradation and persist in the environment for a long time. Also excessive and prolonged uses of synthetic insecticides can lead to the development of insect resistance. To overcome the environmental hazards caused by the application of synthetic insecticides, biopesticides offer a better alternative. For instance, Bacillusthuringiensis accounts for 90% of the bio insecticide market; it produces insecticidal toxins called delta endotoxins (BT toxin) which are proteinaceous, readily biodegradable and also have a short half-life inside the insect midgut. In the recent past, many of the crop pests have developed resistance towards BT toxins and this includes: Ostrinia nubilalis (European corn borer), Heliothis virescens (Tobacco bud worm), Pectonophora gossypiella (Pink bollworm moth), Culex quinquefasciatus (Mosquito), Aedes aegypti (Yellow fever mosquito), Trichloro plusiani (Tiger moth), Leptinotarsa decemlineata (Colorado potato beetle), Spodoptera exigua (Beet armyworm), Spodoptera littoralis(Egyptian cotton leaf worm), and Chryosomela scripta(Cottonwood leaf beetle)3-7. Therefore, insecticidal toxins of Photorhabdus luminescens can be considered as a potential substitute to the BT toxin. Photorhabdus luminescens is a gram-negative entomopathogenic enterobacterium that exists in mutualistic symbiosis with nematodes of the family Heterorhabditidaefound in the gut of infective insect host Heterorhabditis bacteriophora. After entering inside the insect host, the nematode releases the bacteria by regurgitation directly into the insect's hemocoel. In the hemocoel, these bacteria replicate rapidly and cause lethal sepsis by producing different high molecular weight toxins that kill the insect within 48–72 hours. Among various insecticidal toxins produced by the Photorhabdus luminescens purified Tca was shown to disrupt the insect midgut epithelium in a manner similar to the endotoxins from Bacillus thuringiensis. There are several studies reporting that glycosyl-phosphatidyl-inositol (GPI) anchored aminopeptidase N (APN)10 and cadherin-like protein11 of Manducasexta act as receptors for the BT toxins. Among these two receptors, the APN receptor belongs to the Zn-binding metalloprotease family. The C-terminal region of the APN binding site is rich in N-acetylgalactosamine (GalNAc) and acts as the binding site of the Cry1Ac toxin10,12. Although the toxin protein produced by Photorhabdusbacterium has been proved to be toxic against wide variety of insects, structural information of these proteins is yet to be resolved through X- ray / NMR experiments. Therefore in the present study we report 3D models for TcaA toxin Photorhabdus luminescens w14)as well as APN receptor Helicoverpa armigera. Further we have also examined the molecular interaction of TcaA toxin with the APN receptor through the docking studies.Material and MethodsPhylogenetic analysis: Amino acid sequence of TcaA from Photorhabdus luminescens in FASTA format along with the insecticidal cry toxins from Bacillus thuringiensis such as 1DLC_A13, 1JI6_A 14, 3EB715, 1CIY_A16, 2C9K_A17, Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 2(2), 40-49, February (2013) Res. J. Recent Sci. International Science Congress Association 41 1W99_A18 and 1I5P_A19 were submitted to the CLUSTALW20. Output generated by CLUSTALW tool was saved as ‘allign.phy’. Then the CONSENSE tree for 100 data sets was generated with the help of Phylip21. Molecular modeling of TcaA toxin from Photorhabdus luminescens and Aminopeptidase N from Helicoverpa armigera.TcaA toxin sequence from Photorhabdus luminescens was submitted to ProDom22 server for detecting the presence of any domains. Templates selection for TcaA and APN were based on the output generated by PSI-BLAST tool23. To predict the tertiary structure, primary amino residues of these two proteins along with the template suggested by PSI-BLAST tool were submitted to SWISS-MODEL sever24 in the alignment mode. As an alternative to homology modeling, threading method was also attempted by accessing the I-TASSER server25. The initial 3D models of the toxins as well as APN were subjected to loop refinement based on the output generated by ERRAT26, ProSA27and PROCHECK28 server. Loop regions of the proteins showing high percentage of error were further refined through ModLoop server29. After re-validating the refined structures, their structural homologs were searched using the services of DaliLite server30. Docking studies: Tertiary structures of all domains of TcaA were taken for docking studies with APN receptors from Helicoverpa armigera using Hex software31. Docked conformations and interaction energies were recorded at the end of the docking exercise. During the dock operation, the total energies were calculated based on shape as well as electrostatics using a default grid spacing of 0.6 . Results and DiscussionPhylogenetic analysis: Phylogenetic analysis of TcaA from Photorhabdus luminescens was done by including similar group of toxin sequences from other organisms along with Cry toxins of Bacillus thuringiensis. The consense tree generated for TcaA toxin branched into two distinct groups, wherein the first group consisted of only Cry toxins from B. thuringiensis, while the secondgroup comprised of only TcaA toxins from different species. Further, based on the location of TcaA sequence of P. luminescens clade in the consensus tree, it is possible to deduce that, the sequence was evolutionary unrelated to the remaining Cry toxins of Bacillus thuringiensis; however, it showed close evolutionary relationship with TcaA toxin of Burkholderia rhizoxinica (figure-1). Molecular modeling studies: The results obtained from ProDom server suggest that TcaA sequence from Photorhabdus luminescens has six domains (table-1). Although, search for template sequences from PDB using PSI Blast tool did not yield any sequence homologs for any of these 6 domains of TcaA, homologous sequences for the receptor sequence “aminopeptidase N” (APN) from Helicoverpa armigera could be retrieved from PDB. Among the various template sequences reported by the PSI Blast tool, human endoplasmic reticulum aminopeptidase which had the maximum sequence identity (39%) was selected for modeling APN from H. armigera (table-2). Figure-1 Consensus tree generated for TcaA from Photorhabdus luminescens using Promlk program of PHYLIP Table-1 Detection of domains in TcaA sequence from Photorhabdus luminescens using the ProDom server Position ProDom domain Score E value 1-117 #PD287783 615 5e-63 118-224 #PD156536 555 5e-56 225-595 #PD321543 1989 0 596-836 #PD185778 1296 6e-142 860-999 #PDA1W468 741 1e-77 1000-1095 #PD230859 405 1e-38 The 3D structure of APN from H. armigera generated by SWISS MODEL server displayed four domains. While the first (D-I) and third domain (D-III) arranged in sandwich form, consisted of -sheets (D- I=16; D - III= 6), the fourth (D-IV) domain is made up of -helices (D-IV=15). The second domain (D-II) had both -helices (10) and -sheets (5). Except for minor differences in D-I and D-II, same number of helices / sheets were present in the corresponding domains of the PDB template, 3MDJ_A. Comparison of homology modeled APN structure of H. armigera with crystal structure of crystal structure of human aminopeptidase (3MDJ_A), shows that overall topology of all the four domains (D I, II, III and IV) of the predicted models appears to have more or less similar orientation to that of 3MDJ_A. The second domain (D- II) is having a good sequence homology with the corresponding domain of the crystal structure of human aminopeptidase (3MDJ_A), a catalytic domain determined by Nguyen etal. (2011). Therefore, it is possible to speculate similar activity for the theoretical structure of D-II of APN. Sequence analysis revealed that, except for the residue T in the motif “323GATEN327, remaining residues followed by the motif “359HExxHx18E382” of D-II was well conserved with human aminopeptidase 3MDJ_A (figure-2). Methionine located at 267th position of 3MDJ_A was replaced with threonine at the corresponding position of D-II. Studies carried out byNguyen etal. on human aminopeptidase have clearly shown that while “265GAMEN269” motif plays roles in Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 2(2), 40-49, February (2013) Res. J. Recent Sci. International Science Congress Association 42 the exopeptidase specificity of aminopeptidase through interaction with the N-terminal amino acid of the substrate, the motif region “301HEXXH324“ functions as receptors for zinc ligands and are essential for the catalytic activity of the enzyme . Table-2 Summary of (A) PDB templates generated by PSI blast results and (B) sequence alignment of APN sequence from Helicoverpa armigera at the end of 20th iteration (A) B) Query 40 RLPEDLDPINYVVEVTPYFTATDTKEAFTFDGLVTITLRTLKADLNALIIQENVRTINSV 99 RLPE + P++Y + + T TF G + + T + +I+ + I+ Sbjct 17 RLPEYVIPVHYDLLIHANLTTL------TFWGTTKVEI-TASQPTSTIILHSHHLQISRA 69 Query 100 ALTTEAGTSVPLHATTPFERITAYHFLKVNLPAGATLENGAVYKLTVDYVGNINETPLSR 159 L AG + P + + ++ L A L G Y + + Y GN++ET Sbjct 70 TLRKGAGERLS---EEPLQVLEHPRQEQIALLAPEPLLVGLPYTVVIHYAGNLSET--FH 124 Query 160 GVFRGSHKDANGNTRWYAATHLQPTNSRQAFPSFDEPGFKSTFDIIINRPVTFAPSFSNM 219 G ++ +++ G R A+T +PT +R AFP FDEP FK++F I I R + Sbjct 125 GFYKSTYRTKEGELRILASTQFEPTAARMAFPCFDEPAFKASFSIKIRREPRHLAISNMP 184 Query 220 GIKSSDLVNNRIREVFYTTPRMSAYLVTFHISEDFTVIANNNNDARSYRILARPTAAGQG 279 +KS + I + F T +MS YLV F IS+ F ++ + A P Q Sbjct 185 LVKSVTVAEGLIEDHFDVTVKMSTYLVAFIISD-FESVSKITKSGVKVSVYAVPDKINQA 243 Query 280 QYALEVGPPVTNWLGEYLGIDYYSMDENTNMKNDQIASPYWASGATENWGLVTYRELRLL 339 YAL+ + + +Y I Y K D A P + SGA ENWGL TYRE LL Sbjct 244 DYALDAAVTLLEFYEDYFSIPY------PLPKQDLAAIPDFQSGAMENWGLTTYRESALL 297 Query 340 YQEGETNALDKMYIGTITAHELAHKWFGNLITCRWWDNVWINEGFASYFEYFAMDGVDKT 399 + +++A K+ I AHELAH+WFGNL+T WW+++W+NEGFA + E+ ++ Sbjct 298 FDAEKSSASSKLDITMTVAHELAHQWFGNLVTMEWWNDLWLNEGFAKFMEFVSVSVTHPE 357 Query 400 MELEDQFNIMYVQSALSADATLSTRALQHTVNSPTEVTGHFSGISYSKGASLLLMLKHFL 459 +++ D F A+ DA S+ + V +P ++ F +SY KGA +L ML+ +L Sbjct 358 LKVGDYFF-GKCFDAMEVDALNSSHPVSTPVENPAQIREMFDDVSYDKGACILNMLREYL 416 Query 460 TENTFKKALNIFLEARKFEHAFPADLFSAFATAVQQDGVP-------------------S 500 + + FK + +L+ +++ DL+ + A+ DGV Sbjct 417 SADAFKSGIVQYLQKHSYKNTKNEDLWDSMASICPTDGVKGMDGFCSRSQHSSSSSHWHQ 476 Query 501 NTFDIASFMKYWVEEPGYPVLEVSVNSAAGRIELSQKRFLVSATATP-TDQVWPLPLTYT 559 D+ + M W + G+P++ ++V + + Q+ ++ + P T +W +PLT+ Sbjct 477 ERVDVKTMMNTWTLQRGFPLITITVRGR--NVHMKQEHYMKGSDGAPDTGYLWHVPLTFI 534 Query 560 TESNPDWQNLLPSKVMTAKTDFIERNVGTNEWVIFNVQQKGIYRVNYDTRNWELLAAALS 619 T + +++ ++ KTD + EW+ FNV G Y V+Y+ W+ L L Sbjct 535 TSKS----DMVHRFLLKTKTDVLILPEEV-EWIKFNVGMNGYYIVHYEDDGWDSLTGLLK 589 Query 620 RDHTAIHHLNRAQIVDDVFALMRSGQITYRLGFKVLDFLKKDTSYYSWYPAITGFNWLRN 679 HTA+ +RA ++++ F L+ G+++ + +LK +T P G N L Sbjct 590 GTHTAVSSNDRASLINNAFQLVSIGKLSIEKALDLSLYLKHETE---IMPVFQGLNELIP 646 Query 680 RF-LHLPTTLAAFDEILYGFLDAVITDL-GYDVVANE-PLTRTLNRFFTLSFACNIGHKG 736 + L + + FL ++ DL +E ++ + R L AC ++ Sbjct 647 MYKLMEKRDMNEVETQFKAFLIRLLRDLIDKQTWTDEGSVSERMLRSELLLLACVHNYQP 706 Query 737 CVDNAVQKFVALKDNSV--AVNPNLRRHVFCEGLRAGGLDEWQYLYNRRQASNNQGDEVA 794 CV A F K+++ ++ ++ VF G A + W +LY++ Q S + ++ Sbjct 707 CVQRAEGYFRKWKESNGNLSLPVDVTLAVFAVG--AQSTEGWDFLYSKYQFSLSSTEKSQ 764 Query 795 MLRSLGCTSNTAAGQAYLKMILDDDVVKAQDRVNAFSFFYMGHRDNAKAGLQFLKDNVDA 854 + +L T N Q L D +K Q+ + QFL+ N + Sbjct 765 IEFALCRTQNKEKLQWLLDESFKGDKIKTQE-FPQILTLIGRNPVGYPLAWQFLRKNWNK 823 Query 855 IRKAVVLPAWFNN--VLTTTAGYLDEAGLRDME---EWLLANQNAVPEFAVGISAITSAR 909 + + L + V+ TT + L +++ L N + + I I Sbjct 824 LVQKFELGSSSIAHMVMGTTNQFSTRTRLEEVKGFFSSLKENGSQLRCVQQTIETI---E 880 Query 910 NNMQWGSDNAATI---IAAANDEDPPEDGGSGEE 940 N+ W N I + + E PE +G E Sbjct 881 ENIGWMDKNFDKIRVWLQSEKLEHDPEADATGLE 914 Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 2(2), 40-49, February (2013) Res. J. Recent Sci. International Science Congress Association 43 Figure-2 Multiple sequence alignment of Aminopeptidase N from H. armigera with selected PDB homologs using ClustalW. 2YD0_A --------------------------------------- R LP E Y VIPV HY 11 3QNF_A --------------------------------------- R LP E Y VIPV HY 11 3MDJ_A --------------------------------------- R LP E Y VIPV HY 11 3SE6_A --------------------------------------- R LP S VVIPL HY 11 3Q7J_A -------------------------------------------- M E V E K Y 6 gi|33641859|gb|AAQ24379.1| M Q FI T IILLA S AAII S A D FPLPP E F DE P E FF STS D P D SY R LP ED L D PI NY 50 : :* 2YD0_A D LLI H A N L TT L T FW G -------- TT K V E I T A SQ P TST IIL HSHH L Q I S R A 53 3QNF_A D LLI H A N L TT L T FW G -------- TT K V E I T A SQ P TST IIL HSHH L Q I S R A 53 3MDJ_A D LLI H A N L TT L T FW G -------- TT K V E I T A SQ P TST IIL HSHH L Q I S R A 53 3SE6_A D LFV H P N L TS L D FVA -------- S E K I E VLV SN A TQ FIIL HS K D L E I TN A 53 3Q7J_A D L T L D F D I Q KR T F NG -------- T E T I T A D A G D ---- IVL D AV G L Q I N WM 44 gi|33641859|gb|AAQ24379.1| VV E V T P Y F T A T D T K E AF T F D G LV T I T L R T L K A D L N ALII Q E N V R T I NS VA 100 : : : : .: .: *: . . 2YD0_A T L RK G A G E R L S E -- E PL Q VL E H P R Q E Q IALLAP E PLLV G LP YT VVI HY A G 101 3QNF_A T L RK G A G E R L S E -- E PL Q VL E H P R Q E Q IALLAP E PLLV G LP YT VVI HY A G 101 3MDJ_A T L RK G A G E R L S E -- E PL Q VL E H P R Q E Q IALLAP E PLLV G LP YT VVI HY A G 101 3SE6_A T L QS EED S R Y M K P G K E L K VL SY PA H E Q IALLVP E K L T P H L K YY VAM D F Q A 103 3Q7J_A K V NG R D T AF TY D G ---- QT V R AP G D S ------------- Q P Q K I E I S FA G 77 gi|33641859|gb|AAQ24379.1| L TT E A GTS VPL H A TT PF E R I T A YH FL K V N LPA G A T L E NG AV Y K L T V D Y V G 150 . : : : : : . 2YD0_A N L S E T -- F HG F Y K STY R T K E G E L R ILA STQ F E P T AA R MAFP C F DE PAF K A 149 3QNF_A N L S E T -- F HG F Y K STY R T K E G E L R ILA STQ F E P T AA R MAFP C F DE PAF K A 149 3MDJ_A N L S E T -- F HG F Y K STY R T K E G E L R ILA STQ F E P T AA R MAFP C F DE PAF K A 149 3SE6_A K L G D G -- F E G F Y K STY R T L GG E T R ILAV T D F E P TQ A R MAFP C F DE PLF K A 151 3Q7J_A K V S D S -- L SG I YY A G R E NG ------ MI TTH F Q A T D A RR MFP C V D H PA Y K A 119 gi|33641859|gb|AAQ24379.1| N I N E T PL S R G VF R GSH K D A NGNT R W Y AA TH L Q P TNS R Q AFP S F DE P G F K S 200 ::.: *.: . . *.::.* :* **..*.* :*: 2YD0_A S F S I K I RR E P R H LAI SN MPLV K S V T VA E G LI ED H F D V T V K M STY LVAFII 199 3QNF_A S F S I K I RR E P R H LAI SN MPLV K S V T VA E G LI ED H F D V T V K M STY LVAFII 199 3MDJ_A S F S I K I RR E P R H LAI SN MPLV K S V T VA E G LI ED H F D V T V K M STY LVAFII 199 3SE6_A N F S I K I RR E S R H IAL SN MP K V K T I E L E GG LL ED H F E TT V K M STY LVA Y IV 201 3Q7J_A VFAI T VVI D K D Y D AI SN MPP KR I E V S E R -- K VV E F Q D T P R M STY LL Y V G I 167 gi|33641859|gb|AAQ24379.1| T F D III N R PV T FAP S F SN M G I K SS D LV NN R I R E VF YTT P R M S A Y LV T F H I 250 * * : . . . : * * :**:**: : 2YD0_A S D - F E S V S K I T K SG V K V S V Y AVP D K I NQ A D Y AL D AAV T LL E F Y ED Y F S IP 248 3QNF_A S D - F E S V S K I T K SG V K V S V Y AVP D K I NQ A D Y AL D AAV T LL E F Y ED Y F S IP 248 3MDJ_A S D - F E S V S K I T K SG V K V S V Y AVP D K I NQ A D Y AL D AAV T LL E F Y ED Y F S IP 248 3SE6_A C D - F HS L SG F TSSG V K V S I Y A S P D KR NQTHY AL Q A S L K LL D F Y E K Y F D I Y 250 3Q7J_A G K - F R Y E Y E K Y R --- D I D LILA S L K D I R S K Y PL D MA RK S V E F Y E NY F G IP 213 gi|33641859|gb|AAQ24379.1| S ED F T VIA NNNN D A R SY R ILA R P T AA GQGQY AL E V G PPV TN WL G E Y L G I D 300 . * . : . : .*.*: . :: .*:.* 2YD0_A Y PLP K Q ------ D LAAIP D F QSG AM E N W G L TTY R E S ALLF D A E K SS A SS K 292 3QNF_A Y PLP K Q ------ D LAAIP D F QSG AM E N W G L TTY R E S ALLF D A E K SS A SS K 292 3MDJ_A Y PLP K Q ------ D LAAIP D F QSG AM E N W G L TTY R E S ALLF D A E K SS A SS K 292 3SE6_A Y PL S K L ------ D LIAIP D FAP G AM E N W G LI TY R E TS LLF D P K TSS A S D K 294 3Q7J_A Y ALP K M ------ H LI S VP E F G A G AM E N W G AI T F R E I Y M D IA E N - S AV T V K 256 gi|33641859|gb|AAQ24379.1| YYS M DE NTN M K N D Q IA S P Y WA SG A T E N W G LV TY R E L R LL YQ E G E TN AL D K 350 * . . : * : .** **** *:** : : . * 2YD0_A L G I T M T VA H E LA HQ WF GN LV T M E WW N D LWL N E G FA K FM E FV S V S V TH P E L 342 3QNF_A L G I T M T VA H E LA HQ WF GN LV T M E WW N D LWL N E G FA K FM E FV S V S V TH P E L 342 3MDJ_A L D I T M T VA H E LA HQ WF GN LV T M E WW N D LWL N E G FA K FM E FV S V S V TH P E L 342 3SE6_A LWV T R VIA H E LA HQ WF GN LV T M E WW N D IWL N E G FA K Y M E LIAV N A TY P E L 344 3Q7J_A R NS A T VIA H E IA HQ WF G D LV T M K WW N D LWL N E S FA T FM SY K T M D T LFP E W 306 gi|33641859|gb|AAQ24379.1| M Y I GT I T A H E LA H K WF GN LI TC R WW D N VWI N E G FA SY F E Y FAM D G V D K T M 400 ***:**:***:*:* .**:::*:**.**.::. ::. 2YD0_A K V G - D Y FF G K C F D AM E V D AL NSSH PV ST PV E N PA Q I R E MF DD V SY D K G A C 391 3QNF_A K V G - D Y FF G K C F D AM E V D AL NSSH PV ST PV E N PA Q I R E MF DD V SY D K G A C 391 3MDJ_A K V G - D Y FF G K C F D AM E V D AL NSSH PV ST PV E N PA Q I R E MF DD V SY D K G A C 391 3SE6_A Q F D - D Y FL N V C F E VI T K D S L NSS R PI S K PA E T P TQ I Q E MF DE V SYN K G A C 393 3Q7J_A S FW G D FFV S R TSG AL R S D S L K NTH PI E V D V R D P DE I SQ IF DE I SYG K G A S 356 gi|33641859|gb|AAQ24379.1| E L ED Q F N IM Y V QS AL S A D A T L ST R AL QHT V NS P T E V TGH F SG I SYS K G A S 450 .. :: . .: *: .::.:. .. * :: *. :**.***. 2YD0_A IL N ML R E Y L S A D AF K SG IV QY L Q K HSY K NT K N ED LW D S MA S I C P T D G V K G 441 3QNF_A IL N ML R E Y L S A D AF K SG IV QY L Q K HSY K NT K N ED LW D S MA S I C P T D G V K G 441 3MDJ_A IL N ML R E Y L S A D AF K SG IV QY L Q K HSY K NT K N ED LW D S MA S I C P T D G V K G 441 Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 2(2), 40-49, February (2013) Res. J. Recent Sci. International Science Congress Association 44 3SE6_A IL N ML K D FL G EE K F Q K G II QY L KK F SY R N A K N DD LW SS L SNSC L E S D F TS 443 3Q7J_A IL R MI ED Y A GY EE F RK G I S K Y L N D H K F GN A E GS D LW T AI ED V S ------- 399 gi|33641859|gb|AAQ24379.1| LLLML K H FL T E NT F KK AL N IFL E A RK F E H AFPA D LF S AFA T AV QQ D G VP S 500 :* *:..: : *:..: :*: .: :: **: :: 2YD0_A M D G F CS R SQHSSSSSH W HQ E G V D V K T MM NT W T L Q K G FPLI T I T V R G R N -- 489 3QNF_A M D G F CS R SQHSSSSSH W HQ E G V D V K T MM NT W T L Q K G FPLI T I T V R G R N -- 489 3MDJ_A M D G F CS R SQHSSSSSH W HQ E R V D V K T MM NT W T L Q R G FPLI T I T V R G R N -- 489 3SE6_A GG V CHS D P K M TSN MLAFL G E N A E V K E MM TT W T L Q K G IPLLVV K Q D GCS -- 491 3Q7J_A -------------------- G K PV KR VM E Y WI K N P GY PVI K L KR NG RK -- 427 gi|33641859|gb|AAQ24379.1| N ------------------- T F D IA S FM K Y WV EE P GY PVL E V S V NS AA G R 531 : .* * : * *:: :. . 2YD0_A V H M K Q E HY M K GS D ------ G AP D TGY LW H VPL T FI TS K S ---- D MV H R FL 529 3QNF_A V H M K Q E HY M K GS D ------ G AP D TGY LW H VPL T FI TS K S ---- D MV H R FL 529 3MDJ_A V H M K Q E HY M K GS D ------ G AP D TGY LW H VPL T FI TS K S ---- D MV H R FL 529 3SE6_A L R L QQ E R FL QG VF Q ED P E W R AL Q E R Y LW H IPL TYSTSSS ---- N VI H R H I 537 3Q7J_A I T M YQT R FLL NG E ---------- EE G R WPVPV N I KKK ------ D G V E R IL 461 gi|33641859|gb|AAQ24379.1| I E L SQ KR FLV S A T ------- A T P T D Q VWPLPL TYTT E SN P D W QN LLP S K V 574 : : * ::: . * :*:. .. : : : 2YD0_A L K T K T D VLIL - P EE V E WI K F N V G M NGYY IV HY EDD G W D S L TG LL K GTHT A 578 3QNF_A L K T K T D VLIL - P EE V E WI K F N V G M NGYY IV HY EDD G W D S L TG LL K GTHT A 578 3MDJ_A L K T K T D VLIL - P EE V E WI K F N V G M NGYY IV HY EDD G W D S L TG LL K GTHT A 578 3SE6_A L K S K T D T L D L - P E K TS WV K F N V D SNGYY IV HY E GHG W D Q LI TQ L NQNHT L 586 3Q7J_A L EDE A S ----- I E A D G LI K I N A D S A G F Y R VL Y DD A T F S D VM GHY R ---- D 502 gi|33641859|gb|AAQ24379.1| M T A K T D FI E R N V GTN E WVIF N V QQ K G I Y R V NY D T R N W E LLAAAL S R D HT A 624 : ::. : :*. * * * *: :. : 2YD0_A V SSN D R A S LI NN AF Q LV S I G K L S I E K AL D L S L Y L K H E T E IMPVF QG L N E L 628 3QNF_A V SSN D R A S LI NN AF Q LV S I G K L S I E K AL D L S L Y L K H E T E IMPVF QG L N E L 628 3MDJ_A V SSN D R A S LI NN AF Q LV S I G K L S I E K AL D L S L Y L K H E T E IMPVF QG L N E L 628 3SE6_A L R P K D R V G LI H D VF Q LV G A G R L T L D K AL D M TYY L QH E TSS PALL E G L SY L 636 3Q7J_A L S PL D R I G LV DD LFAFLL SGH I D P E TY R Q R I R N FF DDED HN VI T AIV GQ M 552 gi|33641859|gb|AAQ24379.1| I HH L N R A Q IV DD VFALM R SGQ I TY R L G F K VL D FL KK D TSYYS W Y PAI TG F 674 : :* ::.: * :: *:: . : .: . : : 2YD0_A IPM Y K LM E KR D M N E V E TQ F K AFLI R LL R D LI D K Q -- T W T DE GS V S E R ML R 676 3QNF_A IPM Y K LM E KR D M N E V E TQ F K AFLI R LL R D LI D K Q -- T W T DE GS V S E R ML R 676 3MDJ_A IPM Y K LM E KR D M N E V E TQ F K AFLI R LL R D LI D K Q -- T W T DE GS V S E R ML R 676 3SE6_A E S F YH MM D RR N I S D I S E N L KR Y LL QY F K PVI D R Q -- S W S D K GS VW D R ML R 684 3Q7J_A E Y L R ML T -- H AF DDD A R AF C R S R M Q FL TG K Q DE N -- L K IAL G R V S R ---- 594 gi|33641859|gb|AAQ24379.1| N -- WL R N R FL H LP TT LAAF DE IL YG FL D AVI T D L GY D VVA N E PL T R T L N R 722 : : : : 2YD0_A SQ LLLLA C V HNYQ P C V Q R A E GY F RK W K E SNGN L S LPV D V T LAVFAV G A QS 726 3QNF_A SQ LLLLA C V HNYQ P C V Q R A E GY F RK W K E SNGN L S LPV D V T LAVFAV G A QS 726 3MDJ_A S E LLLLA C V HNYQ P C V Q R A E GY F RK W K E SNGN L S LPV D V T LAVFAV G A QS 726 3SE6_A S ALL K LA C D L NH AP C I Q K AA E LF SQ WM E SSG K L N IP T D VL K IV YS V G A QT 734 3Q7J_A ------ L Y VMV DE SY A EE M S K LF K D F D S A E P ------ E M R SS IA T A Y ALV 632 gi|33641859|gb|AAQ24379.1| FF T L S FA CN I GH K GC V D N AV Q K FVAL K D NS VAV N P N L RR H VF C E G L R A GG 772 :. * . . * 2YD0_A T E G W D FL YS K YQ F S L SST E K SQ I E FAL C R TQN K E K L Q WLL DE S F K G D K I K 776 3QNF_A T E G W D FL YS K YQ F S L SST E K SQ I E FAL C R TQN K E K L Q WLL DE S F K G D K I K 776 3MDJ_A T E G W D FL YS K YQ F S L SST E K SQ I E FAL C R TQN K E K L Q WLL DE S F K G D K I K 776 3SE6_A T A G W NY LL E QY E L S M SS A E QN K IL Y AL STS K HQ E K LL K LI E L G M E G K VI K 784 3Q7J_A TG D L K G LL E K F R S V D R DED R V R II S AF G K L K SNT D L ST V YG MV E K T E I KK 682 gi|33641859|gb|AAQ24379.1| L DE W QY L YN RR Q A SNNQG DE VAML R S L GCTSNT AA GQ A Y L K MIL DDD VV K 822 . * .: . . :. : :: . . . * 2YD0_A TQ - E FP Q IL T LI G R N PV GY PLAW Q FL RK N W N K LV Q K F E L GSSS IA H MVM G 825 3QNF_A TQ - E FP Q IL T LI G R N PV GY PLAW Q FL RK N W N K LV Q K F E L GSSS IA H MVM G 825 3MDJ_A TQ - E FP Q IL T LI G R N PV GY PLAW Q FL RK N W N K LV Q K F E L GSSS IA H MVM G 825 3SE6_A TQ - N LAALL H AIA RR P K GQQ LAW D FV R E N W TH LL KK F D L GSY D I R MII SG 833 3Q7J_A Q D - MI S FF SS AL E T LP ----- G R E FIFA N L D R II R LVI ------------ 714 gi|33641859|gb|AAQ24379.1| A Q D R V N AF S FF Y M GH R D N A K A G L Q FL K D N V D AI RK AVVLP -- AWF NN VL T 870 : . : . :*: * : : . 2YD0_A TTNQ F ST R T R L EE V K G FF SS L K E NGSQ L R C V QQT I E T I EE N I G WM D K N F D 875 3QNF_A TTNQ F ST R T R L EE V K G FF SS L K E NGSQ L R C V QQT I E T I EE N I G WM D K N F D 875 3MDJ_A TTNQ F ST R T R L EE V K G FF SS L K E NGSQ L R C V QQT I E T I EE N I G WM D K N F D 875 3SE6_A TT A H F SS K D K L Q E V K LFF E S L E A QGSH L D IF QT VL E T I T K N I K WL E K N LP 883 3Q7J_A -------------------------------------------------- gi|33641859|gb|AAQ24379.1| TT A GY L DE A G L R D M EE WLLA NQN AVP E FAV G I S AI TS A R NN M Q W GS D N AA 920 Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 2(2), 40-49, February (2013) Res. J. Recent Sci. International Science Congress Association 45 A large cavity was observed between D-II and D-IV of APN model which had resemblance to the template 3MDJ_A Based on this, it is hypothesized that this cavity could provide an easy access to the catalytic site for substrates and might also represent binding site for peptide based substrates32. The D-III is the smallest among all the domains and act as a connecting domain between D-II and D-IV (Figure-3A). Superimposition of backbone of predicted model onto 3MDJ_A shows very low RMSD value of 1.1Å. I-TASSER software was successful in predicting the 3D structures for all the six domains of TcaA from Photorhabdus luminescens (Figure-3B). Among these six domains D-I, II, V and VI are purely made up of -helices (D-I=6, D-II=4, D-V=9 and D-VI =5). Helices of D-II are arranged as antiparallel helix-loop-helix motif in a manner similar to that of solution NMR structure of metalloprotein from Escherichiacoli (PDB ID: 2HZ8) determined by Calhoun et al (2008)33. However, both D-III and D-IV had -helices (D-III=11 and D-IV= 15) as well sheets (D III = 5 and D IV= 2). While the folding pattern of D-III is like a horseshoe comparable to that of the crystal structure of ribonuclease inhibitor (PDB ID 2BEX)34, D-IV architecture resembled to a half-doughnut which is identical to the crystal structure of mitochondrial transcription termination factor 3 from human (PDB ID: 3M66)35. Superimposition of Cbackbone of all these structures onto the top PDB template used by I-TASSER showed a very low RMSD (table-3). Table-3 RMSD of all the six domains of TcaA from Photorhabdus luminescens generated by the Dali serverDomains Templates used by I-TASSER RMSD generated by DALI server Domain I 3V42_A 2.2 Domain II 2HZ8_A 1.5 Domain III 2BEX_ A 2.6 Domain IV 3M66_A 1.9 Domain V 2J0O_A 3.1 Domain VI 3KPH_B 3.3 Structure validation: Validation of the theoretical models of TcaA and APN by the structural assessment tools such as ProSA, ERRAT and PROCHECK showed that there was an improvement in the quality of the predicted structure upon loop refinement. Quality factor of the loop refined APN model calculated by ERRAT reached 57.65 from 48.941. The Z score value computed by the ProSA tool for APN model was -8.89. This suggests that the models are lying within the Z score values of native conformational structures. Ramachandran plot generated by PROCHECK indicates that most of the residues from APN model have and angles in the core and allowed regions. This indicates that most of the main-chain conformations of models are consistent with their side-chain conformations. Summary of the Ramachandran plot revealed that, 85.5% of the residues of APN structure were distributed in the core region, followed by 12.8% in the allowed, 1.0% in the general and remaining 0.9% in the disallowed. The overall G-score calculated for APN model was found to be -0.08 which was above the threshold value indicating the predicted model was satisfactory. Improvement in the quality of the structure upon loop refinement was also seen in all the six TcaA domains of Photorhabdus. The and angles of these theoretical models were present in the core and allowed region. Ramachandran plot statistics indicates that the predicted models were of good quality (table-4). Table-4 PROCHECK summary for all the seven domains of TcaA from Photorhabdus luminescens Domains Residues Present in G-factor Core region Allowed region General region Disallowed region I 84.8% 11.4% 1.9% 1.9% -0.05 II 90.8% 9.2% 0.0% 0.0% 0.01 III 68.8% 25.0% 3.1% 3.1% -0.49 IV 84.8% 10.1% 3.2% 1.8% -0.06 V 82.8% 12.5% 3.1% 1.6% -0.18 VI 87.4% 9.2% 1.1% 2.3% 0.15 Docking studies: HEX was successful in docking the homology modeled aminopeptidase receptor of Helicoverpa armigera on to all the six domains of TcaA toxin model of Photorhabdus luminescens. Based on the docking energies for all the six domains of TcaA presented in table 5, the D-I model got docked onto the APN receptor model with the highest dock energy of -792.9 KJ / mol. Visualization of the docked output using DeepView package reveal that a total of 121 residues of both D-I domain and APN model are involved in the interaction (D-I = 59 and APN = 62 residues) (figure-4). Among these 121 residues, maximum residues are non polar in nature (table-6) (figure-5). The docked complex was stabilized by the formation of strong inter-molecular hydrogen bonds. Table-5 Summary of the docking energy of APN from Helicoverpa armigera onto the different domains of TcaA from Photorhabdus luminescens DomainsBinding energy with APN from Helicoverpa armigera I -792.9 kj/mol II -719.0 kj/mol III -304.1 kj/mol IV -274.9 kj/mol V -588.5 kj/mol VI -656.8 kj/mol Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 2(2), 40-49, February (2013) Res. J. Recent Sci. International Science Congress Association 46 ConclusionBased on the in-silico studies conducted on the interaction of TcaA toxin from Photorhabdus luminescens with the aminopeptidase N receptor of Helicoverpa armigera we can conclude that TcaA toxin can be considered as a potential biopesticide for controlling the pest population affecting the agricultural crops. Table-6 Summary of residues interacting between DI of TcaA and homology modeled APN receptor from Helicoverpa armigeraAcidic Basic Polar Non Polar DI of TcaA asp30,97,106,108,111, glu63,77,101 lys8,66,81,85, arg36,61,70,75,115 gln6,26,41,60,73,76,89, asn7,64,91, ser10,33,67,90,117, tyr25 val14,28,40,94, met5,113, ile9,34, phe11,109, ala37,71,98,116, gly24,105,107 leu65,68,74,86,89,95,112, trp72, pro78 APN receptor (Helicoverpa armigera) asp780, glu109,390,841 lys812, arg118,289,752,781, his121,394, gln367,779, asn108,806,783, ser113,120,277,396,399,786, thr29, 110,389,362, tyr274,307,400 val54,748,782,814,815 met364,790, ile363,398,810 phe395,787,802,820 ala276,372,778,813,818 gly26,119,278,393,397 leu112,291,751,816 trp130,275,631,842 pro111 ( A ) ( B ) Figure-3 3D structure of (A) APN receptor from Helicoverpa armigera (B) TcaA toxin from Photorhabdus luminescens Domain I Domain III Domain V I Domain V Domain IV Domain II Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 2(2), 40-49, February (2013) Res. J. Recent Sci. International Science Congress Association 47 Figure-4 Docking of D-I of TcaA onto homology modeled aminopeptidase N from Helicoverpa armigera using HEX software (Total energy = –792.9 KJ / mol) Figure-5 Amino acid residues of TcaA of Photorhabdus luminescens (Yellow) interacting with homology modeled aminopeptidase N receptor from Helicoverpa armigera (Red) within 5Å distance. Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 2(2), 40-49, February (2013) Res. J. Recent Sci. International Science Congress Association 48 References 1.Roush R., Can we slow adaptation by pests to insect transgenic crops? In: Parsley GJ (ed) Biotechnology and integrated pest management. CAB International, Willinford. (1996) 2.Poinar G. O, Thomas G. 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