International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 1(8), 7-18, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 7 Molecular Modeling and Docking Studies of PirB Fusion Protein from Photorhabdus LuminescensMaithri S.K., Ramesh K.V.*, Dieudonné Mutangana and Deshmukh Sudha Department of Biotechnology, Center for Postgraduate studies, Jain University, Bangalore – 560011 INDIAAvailable online at: www.isca.in Received 4th September 2012, revised 10th September 2012, accepted 15th September 2012Abstract Genetic engineering of Cry proteins from Bacillus thuringiensis (BT) has resulted in the synthesis of various novel toxin proteins which exhibits increased insecticidal activity and highly specificity towards different insect pests. The present study focused on computational studies on PirB sequence from Photorhabdus luminescens. The consensus tree generated by PHYLIP for the PirB sequence revealed that this toxin sequence does not share any ancestral relationship with other Cry toxins from Bacillus thuringiensis considered in this study. Molecular modeling of PirB was followed by construction of two fusion proteins: Type I (PirB-Cry2AaII-Cry2AaIII) and Type II (PirB-Cry2AaII-Garlic lectin). Comparison of the 3D model of PirB with X-ray structure of N-terminal domain 1I5P_A revealed both the structures shared similar architecture. Validation of the tertiary structure of PirB by the structural assessment tools such as ProSA, ERRAT and PROCHECK suggested that the predicted structure was of reasonable quality. Docking studies carried out onto the cadherin receptor showed that Type II fusion protein had a greater affinity, suggesting the possibility of using this fusion protein as a potential bio-pesticide. Keywords: PirB, fusion proteins, modeling, docking, cry toxins.. Introduction Photorhabdusluminescens, a gram-negative bacterium belonging to the Enterobacteriaceae family, and has been used extensively to control large community of insects. Insecticidal activity is due to the high molecular weight insecticidal toxins produced by this bacterium. Insect resistance towards this bacterial toxin has not been reported to date2,3. The genome of the insect pathogen Photorhabdusluminescenssubsp. laumondiistrain TT01 contains numerous genes predicting toxins, hemolysins, and proteases, which may be important for insect pathogenicity. While the loci plu4093 to plu4092 encodes the protein PirA, plu4437 to plu4436 codes for the PirB protein in Photorhabdus luminescens. The Photorhabdus insect related (Pir) toxins act as binary proteins. Both proteins are necessary for injectable but not for oral activity6,7. Compared to other toxins of Photorhabdus luminescens, PirA shows little similarity to known proteins, whereas PirB shows high sequence homology with N-terminal region of the pore-forming domain of the Cry2A insecticidal toxin, thus making them a putative substitute for Bacillusthuringiensis. PirB also has similarities with a developmentally regulated protein from the beetle Leptinotarsadecemlineatawhich appears to have juvenile hormone esterase (JHE) activity 8,9. There are several studies reporting that glycosylphosphatidyl-inositol (GPI) anchored amino peptidase N (APN) and cadherin-like protein functions as receptors of Cry1A toxins. The APN protein belongs to the Zn-binding metalloprotease family of proteins 10,11. The C-terminal stalk of the APN binding site is rich in N-acetylgalactosamine (GalNAc) and acts as the binding site of the Cry1Ac toxin10,12. The studies conducted by Lee et al clearly show that lectins also exhibit good insecticidal activity and therefore transgenic plants engineered to express lectin confers protection against different insect pests. Lectins are group of sugar-binding proteins which recognizes specific carbohydrate structures and are known to agglutinate various animal cells by binding to their cell- surface glycoproteins and glycolipids. The insecticidal activities of plant lectins against a wide range of insect pests belonging to homoptera, lepidoptera, coleoptera and diptera have been well documented13,14. Construction of fusion proteins offers the possibility of exploring toxins having higher insecticidal properties. For instance, different Bt fusion proteins, such as Cry1Ab–Cry1B, Cry1Ac–Cry1Ab and Cry1Ac–GFP have been synthesized and expressed in different plant systems. Transferring of carbohydrate binding domain-III of Cry1C to Cry1Ab resulted in a fusion protein (Cry1Ab–Cry1C), which was highly toxic to the army worm (Spodoptera exigua), which was earlier resistant to Cry1A toxin15-18. There are several studies conducted in the recent past to demonstrate the development of resistance of insects towards BT toxin19,20. Therefore, it is very much essential to design a novel biopesticide, which can circumvent the current problem faced by agricultural crops. In the present study, an attempt has been made to predict theoretical model of fusion protein using International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(8), 7-18, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 8 PirB structure as one of the substituent to Cry toxin complex (1I5P_A), followed by docking studies with cadherin receptor. Material and MethodsPhylogenetic analysis of PirB from Photorhabdus luminescens: Fasta sequence of PirB along with the insecticidal cry toxins from Bacillus thuringiensis such as 1DLC_A 21, 1JI6_A 22, 3EB7 23 , 1CIY_A 24, 2C9K_A 25 (, 1W99_A 26 and 1I5P_A 27 were submitted to the CLUSTALW 28. Output generated by CLUSTALW tool was saved as ‘allign.phy’. Then SEQBOOT program in Phylip 29 was activated. Outfile of the SEQBOOT was renamed as infile and run in “promlk” by analyzing 100 data sets with 5 times jumble. Then outtree of this was renamed as intree and served as input for CONSENSE program. Molecular modeling of PirB from Photorhabdus luminescens: Based on the sequence identity between the PirB sequence and the PDB template as suggested by the PSI Blast tool 30, tertiary structure of PirB was predicted using homology modeling (SWISS MODEL server 31) or threading (I-TASSER server32) approach. The model quality factor suggested by the ERRAT server33 (was taken into account for selecting the appropriate loop regions for refinement using MOODLOOP server34. Minimization of the model was carried out using the Deep View software35. Structurally homologous proteins were obtained by submitting the coordinated of the loop refined model to DALI server36. The refined models were later validated with PROCHECK 37 and ProSA server38. Insilico fusion protein: Two types of fusion proteins were modelled using computational approach. For all docking exercises, HEX (v6.3)39 was used. In the first type, the N-terminal domain model of PirB (PirBI) was initially docked on to the edited X-ray crystal structure of Cry2Aa (PDB id 1I5P)27which contained the coordinates information only for 2nd and 3rddomain. The second type of fusion protein generated by docking the N terminal domain of PirBI to the 2nd domain of Cry2Aa was once again docked onto the PDB structure of garlic lectin (1KJ1)40. This was done because this protein is reported to have a folding pattern comparable to domain III of Cry toxin2541 and moreover it has insecticidal property42. 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 . The large number of conformations generated for both the fusion proteins (type I and II) by the HEX package were subjected to rigorous screening, using the X-ray crystal structure of Cry2Aa (1I5P) as the reference, for deciding on the correct orientation of the docked complexes. Docking studies with cadherin receptors: The two fusion proteins generated through HEX were docked onto the X-ray crystal structure of cadherin receptor from Drosophilamelanogaster (PDB ID 3UBH)43 to evaluate the docking affinity. Among the 100 different orientations that were generated by HEX, the best one was selected based on total dock energy. Results and DiscussionThe templates that were selected to generate the consensus tree for PirB from Photorhabdus luminesces were Cry toxins from Bacillus thuringiensis. The output shows PirB toxin sequence as an out group in the tree, suggesting this sequence is evolutionary unrelated to the remaining Cry toxins Bacillus thuringiensis (figure-1). For the PirB sequence for Photorhabdusluminescens, the PSI BLAST tool was able to retrieve wide array of toxins present in diverse group of organisms from protein data bank (table-1 A). Among these toxins structures, the crystal structure of N-terminal domain of cry2Aa (1I5P) was selected as the PDB template for modeling purpose as this template sequence got aligned to the PirB sequence of Photorhabdus luminescens with maximum sequence identity (22%) compared to other toxin sequences (table-1 B). Homology modeling usually starts by searching the database of known protein structures such as protein data bank. Sometimes, the availability of many sequences related to the target makes it necessary to do more sensitive searching with profile methods and Hidden Markov Models 44-46. Fiser and Sali (2000) have recommended the use of PSI BLAST tool 30 as one of the methods to construct a profile from a multiple alignment of the highest scoring hits from an initial BLAST search. Submission of the PirB sequence threaded onto the PDB template 1I5P to the SWISS MODEL server failed in generating a 3D model based on homology modeling approach. However, I-TASSER server, using the same template structure, was able to generate a 3D model for PirB. All the top 10 PDB templates fetched by the server were cry toxin structures displaying sequence similarity to the PirB toxin sequence, which included 1I5P_A also as one of the template (figure-2b). Sequence alignment report generated by I-TASSER output showed that glu56of PirB is well conserved with rest of the sequences. Alignment report also shows that, gln55 and arg100 of PirB were conserved with all the templates except 1I5P_A, which has tyrosine at the corresponding position. Comparison between PirB and 1I5P_A sequences revealed that serine (ser5,34,101,156,218) was maximally conserved followed by glu57,152,215, asn47, 104, 180, val11,15, leu36,129, ile52,219, phe117,132, tyr182,216, pro42, gln80, met132, asp151 and trp220. Among these residues, glu215 and tyr216 of PirB sequence are of significance, because same type of residues are present at the active site of the crystal structure of Cry2Aa-I (domain I) and are involved in binding to Cry2Aa-II (domain II; 1I5P_A). Comparison of the 3D model of PirB with X-ray structure of N-terminal domain 1I5P_A revealed, both the structures shared International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(8), 7-18, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 9 similar architecture (figure-3). Orientation of all the seven helices of PirB model closely matched with that of template (1I5P_A) structure. In addition to these seven helices, I –TASSER was able to predict additional 2 helices (197 - 211 and 122- 126) for PirB model. Further examination of the PirB model and 1I5P_A, revealed that, both of them had common loop segments (designated as L1 to L6) which connected all the seven helices (figure-3). While the number of residues in loop segments L3 and L6 of PirB model and 1I5P_A remained the same (table-2), the number of residues in L1, L2, L4 and L5 varied marginally. However, there was large variation in the number of residues of L5 connecting helix 5 and 6 of PirB and 1I5P_A (table-2). Except pro42 of L2 and ser155 of L6, none of the residues of the loop regions were conserved. The output generated by DALI server revealed that C back bone of the PirB model got super-imposed with top 7 structural homologs (1CIY_A, 1JI6_A, 2QKG_A, 3EB7_A, 2QKGB, 3EB7B, 1I5P_A), belongs to different cry toxins of Bacillus thuringiensis (figure-4). The z-score of these 7 structural homologs varied from 24.3 to 22.4 and RMSD varied from 2.0 to 1.0. Validation of the tertiary structure of PirB 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. Energy minimization of the final PirB model after loop refinement resulted in a decrease of total energy from -4130.422 to -6698.85 KJ / mol. The Z score value computed by the ProSA tool decreased from -7.21 to -7.23 of PirB model and was lying within the Z score values calculated for all experimentally determined protein structures of similar size, deposited in the protein data bank (Figure-5A). Energy profile plot, also generated by the ProSA server, revealed negative energy distribution pattern scored by the amino acid residues of PirB model (Figure-5B), suggesting an improvement in the model quality. The quality factor of the loop refined PirB model calculated by ERRAT reached 74.81 from 57.55. Ramachandran plot analysis performed by PROCHECK tool revealed that 89.4% of the residues of PirB structure were distributed in the core region, followed by 8.0% in the allowed region, 1.0% in the general region and remaining 1.5% in the disallowed region (Table-3). However Ramachandran plot analysis of X-ray crystal structure of 1I5P_A showed that 91.4% of the residues were present in the core, 8.1% of the residues in the allowed region, 0.5% of the residues in the generously allowed region and 0.0% in the disallowed region (table-3B). The overall G-score calculated by the tool for the model was -0.06 which was above the threshold value indicating the predicted model was satisfactory. Kashyap et al (2010) 47 have used similar kind of tools for validating the 3D structureof Cry1Ab17 from Bacillus thuringiensis predicted using X-ray structure of Cry1Aa1 from Bacillus thuringiensis. Although the two fusion proteins, type I (PirBI-Cry2AaII-Cry2AaIII) and II (PirBI-Cry2AaII-Garlic lectin) (figure-6) that were generated using HEX package, displayed similar orientation, type II had better affinity (dock energy = -679.0 KJ / mol) towards the cadherin receptor (figure-7), compared to type I (dock energy = -616.0 KJ / mol). Francis et al. have reported cadherin-like protein functions as a receptor for Cry1A toxins. However, Tanje et al.48 who have modeled “Cry-Garlic lectin” fusion protein using insilico approach, studied the interaction of this toxin protein with aminopeptidase N receptor from Manduca sexta. Efficacy of the fusion protein predicted in the present study cannot be compared with the studies of Tanje et al.48, due to the difference in the type of receptor protein considered. Using PirB in the construction of fusion proteins as one of the component can be beneficial, because of the development of resistance by insects towards the Cry toxins of Bacillusthuringiensis19,20. Residues interacting at the interface of 3 domains of Type II fusion protein model and 1I5P_A: Comparisons were made between the residues interacting at the interface of 3 domains of type II fusion protein model with 1I5P_A (table-4). The results suggest that total number of residues present at the interface of the 3 domains were more in number in case of type II fusion protein (97 residues) compared to 1I5P_A (78 residues). While polar residues were dominant at the interface of type II fusion protein model, on the contrary non- polar types were more in number at the interface of 1I5P_A. Among the 97 residues present at the interface of the type II fusion protein model, 49 of them were associated with PirB model (i. e., domain I) and remaining 48 shared between II and III domain of Type II fusion protein. The residues of domain I that were involved in the interaction with remaining 2 domains of both type II fusion protein model and 1I5P were predominantly polar in nature, with acidic type of residues less in number (table-4). Based on the data generated for residues interacting between Type II fusion protein and crystal structure of cadherin receptor from Drosophilamelnogaster, it is seen that polar residues of Cry2Aa –II (domain II of Type II fusion protein) and non-polar residues of receptor (Cadherin) were maximally involved in the interaction (table-5) (figure-8). Conclusion Molecular modeling studies of PirB toxin resulted in the construction of 2 types of fusion protein, built using the Cry2Aa toxin complex from Bacillusthuringiensis. Among these two types of fusion protein, type II showed better affinity with the Cadherin receptor and therefore can be considered as probable alternative to the currently used BT toxins for controlling the agricultural crop pests. International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(8), 7-18, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 10 Figure-1 Consensus tree generated for PirB from Photorhabdus luminescens using Promlk program of PHYLIP. The number on the branches indicate the number of times the partition of the species into the two sets which are separated by that branch occurred among the trees, out of 100 trees. The tree is an unrooted one. Figure-2 Alignment of N-terminal domain of PirB from Photorhabdus luminescens with the top 10 PDB templates generated by the I-TASSER server International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(8), 7-18, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 11 Figure-3 Tertiary structure of PirB from Photorhabdus luminescens generated by the I-TASSER server. The N –terminal region of PirB was predicted by furnishing the PDB coordinates of the X-ray structure of cry toxin “cry2Aa” from Bacillus thuringiensis in the template option of I-TASSER server Figure-4 Dali server output showing super-imposed structural homologs for PirB from Photorhabdus luminescens Green: PirB; red: 1CIY_A; grey: 1JI6_A; orange: 2QKG_A; dark blue: 3EB7_A; light blue: 2QKGB; green: 3EB7B; yellow: 1I5P_A International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(8), 7-18, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 12 (A) (B) Figure-5 ProSA server output generated for the N-terminal domain of PirB from Photorhabdus luminescens. Graphical display of (A) Z score plot (B) Energy profile plot Figure-6 3D model of fusion protein (Type II) generated by docking PirB model from Photorhabdusluminescens onto the crystal structures of cry2Aa (PDB ID: 1I5P; Bacillusthuringiensis) and garlic lectin (PDB ID: 1KJ1; Allium sativum) using HEX software. Image was generated using SPDV package International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(8), 7-18, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 13 Figure-7 Docking of Type II fusion protein onto X-ray crystal structure of N-cadherin from Drosophila melanogaster (PDB ID 3UHB_A)using HEX software (Total energy = –679.0 KJ / mol)Figure-8 Amino acid residues of Type II fusion protein (Green: PirB; Blue: Cry2Aa-II; Yellow: Garlic lectin ) interacting with crystal structure of cadherin receptor from Drosophila melagaster (Red) within 5 distance International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(8), 7-18, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 14 Table-1 Summary of (A) PDB templates generated by PSI blast results and (B) sequence alignment of PirB sequence from Photorhabdusluminescens at the end of 20th iteration (A)E-value BETTER than threshold Sequences producing significant alignments Score Bits E-Value pdb|1JI6|A Chain A, Crystal Structure Of The Insecticidal Bac... 226 6e-67 pdb|1DLC|A Chain A, Crystal Structure Of Insecticidal Delta-E... 221 2e-65 pdb|3EB7|A Chain A, Crystal Structure Of Insecticidal Delta-E... 213 5e-62 pdb|1I5P|A Chain A, Insecticidal Crystal Protein Cry2aa 201 3e-57 pdb|2IZW|A Chain A, Crystal Structure Of Ryegrass Mottle Viru... 175 2e-51 pdb|1CIY|A Chain A, Insecticidal Toxin: Structure And Channel... 179 7e-50 pdb|1W99|A Chain A, Mosquito-Larvicidal Toxin Cry4ba From Bac 176 7e-49 pdb|2C9K|A Chain A, Structure Of The Functional Form Of The M... 170 1e-46 pdb|2QNT|A Chain A, Crystal Structure Of Protein Of Unknown F... 126 3e-34 pdb|3VDG|A Chain A, Crystal Structure Of Enolase Msmeg_6132 (. 81.4 1e-16 pdb|3VC5|A Chain A, Crystal Structure Of Enolase Tbis_1083(Ta... 77.9 2e-15 pdb|3VA8|A Chain A, Crystal Structure Of Enolase Fg03645.1 (T... 77.1 3e-15 pdb|3S8Y|A Chain A, Bromide Soaked Structure Of An Esterase F... 56.0 1e-08 pdb|3I6Y|A Chain A, Structure Of An Esterase From The Oil-Deg... 55.6 2e-08 pdb|3LS2|A Chain A, Crystal Structure Of An S-Formylglutathio... 50.6 8e-07 (B) ALIGNMENTS �pdb|1I5P|A Chain A, Insecticidal Crystal Protein Cry2aa Length=633 Score = 201 bits (510), Expect = 3e-57, Method: Composition-based stats. Identities = 52/240 (22%), Positives = 100/240 (42%), Gaps = 33/240 (14%) Query 18 AVKTSALEWDVTD--IVKNAIIGGIS--FIPSVGPAI-----SFLVGLFWPQSKENIWEG 68 ++ +EW TD + ++G +S + VG I S L G+ +P N+ + Sbjct 33 TIQKEWMEWKRTDHSLYVAPVVGTVSSFLLKKVGSLIGKRILSELWGIIFPSGSTNLMQD 92 Query 69 IVKQIERMIEE----SALKTIKGILAGDIAYIQERMATVADLLD--KHPGSEEARSAFNN 122 I+++ E+ + + L + L G A I+E V + L+ ++P S+ N Sbjct 93 ILRETEQFLNQRLNTDTLARVNAELIGLQANIREFNQQVDNFLNPTQNPVPLSITSSVNT 152 Query 123 LAENIDGYHKKFNNFSDDVNYQIL--PMFSTTVMMQITYWVAGLERKDEIGLSNIDIEKV 180 + + +F YQ+L P+F+ M +++ + DE G+S + Sbjct 153 MQQLFLNRLPQF----QIQGYQLLLLPLFAQAANMHLSFIRDVILNADEWGISAATLRTY 208 Query 181 RGLIKKTVEQANSYINNIYD---RELNDALNNSTADTVANNVMSVHGHCRLHGIEYISIW 237 R ++ ++Y N Y R LN L ++++ + L+ EY+SIW Sbjct 209 RDYLRNYTRDYSNYCINTYQTAFRGLNTRL---------HDMLEFRTYMFLNVFEYVSIW 259 Table-2 Summary of the loop segments involved in the connectivity of various helices of 3D model of PirB and PDB template 1I5P_A PirB Helices 1I5P_A L1(pro 27 -ala 32 ) 1- 2 L1(asp 45 -pro 52 ) L2(phe 40 -glu 46 ) 2- 3 L2(ser 67 -ile 73 ) L3(glu 61 -leu 65 ) 3- 4 L3(phe 82 -thr 87 ) L4(lys 92 -glu 98 ) 4- 5 L4(asn 102 -asn 106 ) L5(asn 119 -ser 121 ) 5- 6 L5(asn 136 -ser 145 ) L6(gly 154 -ser 156 ) 6- 7 L6(leu 193 -ala 195 ) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(8), 7-18, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 15 Table-3 Summary of the PROCHECK analysis for the N-terminal domain of PirB from (A) Photorhabdus luminescens (B) 1I5P_A from Bacillus thuringiensis (a) (b) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(8), 7-18, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 16 Table-4 Comparison of the interacting residues at the interface of 3 domains of Type II fusion protein model and crystal structure of 1I5P Cry2Aa_II Total Acidic PirBasp88, 111, 122, 183, 188glu46, 50, 81, 108, 172, 185 (types=2; total = 11) asp16, 25 (types=1; total = 2) GL*asp17,35, glu19, 27 (types = 2; total = 4) 17 Cry2Aa_I asp22,45 (types=1, total =2) asp 420 ( types=1; total = 1) Cry2Aa_III asp578,582,589 (types = 1; total =3) 6 Basic PirBarg184, lys45,92,115,116,169 his114 (types=3; total = 7) arg26,32,, lys30,142, his144 (types=3; total =5) GLarg, his22,36 (types=2; total =3) 15 Cry2Aa_I arg9,209,213,217,232,237,245 , his21,239 (types=2; total =9) arg 375 , his 468 (types=2; total = 2) Cry2Aa_III arg548 (types=1; total = 1) 12 PirB GL Polar asn 47,118,119,125,175,179,180,187 , gln43,173, ser44,121,176 thr67,85 , tyr78,113,117 (types=5; total =18) asn 11, gln 27,34 ser 19,21 , thr22,28,37,tyr20 (types=5; total =9) gln 14,26 , ser 15,37 , tyr 11,21,34 (types=3; total =7) 34 Cry2Aa_I asn3,6,221,235 , ser6,7,25,260,266,270,271 thr11,236 , tyr264 (types=4; total=14) asn 274,341,416,469,472,473 , gln 283, 284,286,399 , ser278,280.287,309,363,370,376,415, thr285,364 , tyr417,421 (types=4; total = 22) Cry2Aa_III asn576,577,581 , ser588,621 , thr492,495,573 , tyr475,633 (types=4; total = 10) 22 Non polar PirBala73,77,84, , gly70,74,112, ile68,71,181, phe120, pro42, trp41, val171 ( types=7; total =13) ala29,31 , gly146 , ile13,33, leu18,36, phe23,143,, val9,15,145 ( types=6; total =12) GLala12, gly13, ile24, leu16, pro20, val18 ( types=6; total =6) 31 Cry2Aa_I gly8,272 , leu, met268, trp41 (types=4; total = 5) ala273,277,cys362, gly279,281,310, leu275,308,366,369, phe288,418,422, pro282,367,368,419, val365,374 (types=7; total =19) Cry2Aa_III gly493,579, ile474,484,496,590, leu547,632, met620, phe494,624, pro631, phe494,624 (types=7; total = 14) 38 Note: * GL = Garlic lectinTable-5 Summary of residues interacting between Type II fusion protein and crystal structure of cadherin receptor from Drosophilamelnogaster (PDB ID: 3UHB_A) Acidic Basic Polar Non Polar Type II Fusion protein PirB (Domain I) alu 148,161 ly s150 , arg 164 NILala 221 Cry2Aa-II (Domain II) NIL arg 323,353,360 , his 457 , lys 419 asn 222,251,253,393,417, 420,421 , gln239,249,347, ser252,351,358, thr237, tyr245,254,392ala 238,359 , ile 255, Garlic Lectin (Domain III) asp 456,513 , glu 440,512 NIL tyr 442, ile 523 ,pro 441 , trp 524 , val 439 Cadherin receptor (PDB ID: 3UHB_A) glu 456,539,613,637,638,657,659,791 arg 546,630,631,639,718, 745 , lys492,493,494,619gln 690,692 , ser 634,655,724,786,788 , thr 635,743 , tyr542,656gly 691,787 , ile 671 , leu 548,717 , met 653 , phe632,714, pro540,541,572,636,654,720, val583,615,627,722 International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(8), 7-18, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 17 References 1.Poinar G.O., Thomas G.M. and Hess R., Characteristics of the specific bacterium associated with Heterorhabditis bacteriophora (Heterorhabditidae: Rhabditida), Nematologica.,23, 97–102 (1977) 2.Chattopadhyay A. Bhatnagar N. and Bhatnagar R., Bacterial insecticidal toxins, Crit. Rev. Microbiol.,30, 33–54 (2004)3.Daborn P.J., Waterfield N.R., Silva C.P., Au C.P., Sharma S. and Ffrench-Constant R.H., A single Photorhabdus gene, makes caterpillars floppy (mcf), allows Escherichia coli to persist within and kill insects, Proc. Natl. Acad. Sci USA,99, 10742–10747 (2002)4.Duchaud E et al., The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens, Nat. Biotechnol,21, 1307–1313 (2003)5.Waterfield N.R., Hares M., Yang G., Dowling A. and ffrench-Constant R.H., Potentiation and cellular phenotypes of the insecticidal toxin complexes of Photorhabdus bacteria, Cell Microbiol7, 373–382 (2005 a)6.ffrench-Constant R. H., Dowling A. and Waterfield N. R., Insecticidal toxins from Photorhabdus bacteria and their potential use in agriculture, Toxicon49, 436–451(2007)7.Yang G., Dowling J., Gerike U., ffrench-Contant R. H. and Waterfield N. R., Photorhabdus virulence cassettes confer injectable insecticidal activity against the wax moth, J. Bacteriol188, 2254–2261 (2006)8.Waterfield N. R., Kamita S. G., Hammock B. D. and ffrench-Constant R. H., The Photorhabdus Pir toxins are similar to a developmentally regulated insect protein but show no juvenile hormone esterase activity, FEMS Microbiol.Lett 245, 47–52 (2005 b)9.Wilkinson P., Waterfield N. R., Crossman L., Corton G., Sanchez-Contreras M. et al. Comparative genomics of the emerging human pathogen Photorhabdus asymbiotica with the insect pathogen Photorhabdus luminescens. BMC Genomics10, 302-323 (2009)10.Knight P. J., Crickmore N. and Ellar D. J., The receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase N, Mol. Microbiol11, 429–436 (1994)11.Francis B.R. and Bulla Jr L.A., Further characterization of BT-R1, the cadherin- like receptor for Cry1Ab toxin in tobacco hornworm (Manduca sexta) midgets, Insect Biochem. Mol.Biol, 27, 541–550 (1997)12.Knight P.J., Carroll J. and Ellar D.J., Analysis of glycan structures on the 120 kDa aminopeptidase N of Manduca sexta and their interactions with Bacillus thuringiensis Cry1Ac toxin, Insect Biochem. Mol. Biol34, 101–112 (2004)13.Lee M.K., You T.H., Gould F.L. and Dean D.H., Identification of residues in domain III of Bacillus thuringiensis Cry1Ac toxin that affect binding and toxicity, Appl. Environ. Microbiol65, 4513–4520 (1999)14.Bharathi Y., Reddy T.P., Reddy V.D. and Rao K.V., Plant lectins and their utilization for development of insect resistant transgenic crop plants, in: Pests and Pathogens: Management Strategies, BS Publications, 457–489 (2010)15.Ho N.H., Oliva B.N., Datta K., Frutos R. and Datta S.K., Translational fusion hybrid Bt genes confer resistance against yellow stem borer in transgenic elite vietnamese rice (Oryza sativa L.) cultivars, Crop Sci.46, 781–789 (2006)16.Honee G., Vriezen W. and Visser B., A translation fusion product of two different insecticidal crystal protein genes of Bacillus thuringiensis exhibits an enlarged insecticidal spectrum, Appl. Environ. Microbiol.56, 823–825 (1990)17.Harper B.K., Mabon S.A., Leffel S.M., Halfhill M.D., Richards H.A. and Moyer K.A. et al., Green fluorescent protein as a marker for expression of a second gene in transgenic plants, Nat. Biotechnol17, 1125–1129 (1999)18.de Maagd R.A., Kwa M.S., van der Klei H., Yamamoto T., Schipper N., Vlak J.M. et al., Domain III substitution in Bacillus thuringiensis delta-endotoxin CryIA(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognition, Appl. Environ. Microbiol62, 1537–1543 (1996)19.Hoy M.A., Myths, models and mitigation of resistance to pesticides, Phil. Trans. R. Soc. Lond. B353, 1787–1795 (1998)20.Michaud D., Avoiding protease mediated resistance in herbivorous pests. Trends Biotech 15, 4–6 (1997)21.Li J.D., Carroll J. and Ellar D.J., Crystal structure of insecticidal delta-endotoxin from Bacillus thuringiensis at 2.5 A resolution, Nature,353, 815-821 (1991)22.Galitsky N., Cody V., Wojtczak A., Ghosh D., Luft J. R., Pangborn W. and English L., Structure of the insecticidal bacterial delta-endotoxin Cry3Bb1 of Bacillus thuringiensis, Acta crystallogr D Biol Crystallogr57, 1101-1109 (2001)23.Guo S., Ye S., Liu Y., Wei L., Xue J., Wu H., Song F., Zhang J., Wu X., Huang D. and Rao Z., Crystal structure of Bacillus thuringiensis Cry8Ea1: An insecticidal toxin toxic to underground pests, the larvae of Holotrichiaparallela, J struct Biol , 168, 259-266 (2009)24.Grochulski P., Masson L., Borisova S., Pusztai-Carey M., Schwartz J. L., Brousseau R. and Cygler M., Bacillus thuringiensis CryIA(a) insecticidal toxin: crystal structure and channel formation, J Mol Biol.254, 447-464 (1995) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(8), 7-18, December (2012) Int. Res. J. Biological Sci. International Science Congress Association 18 25.Boonserm P., Mo M., Angsuthanasombat C. and Lescar J., Structure of the functional form of the mosquito larvicidal Cry4Aa toxin from Bacillus thuringiensis at a 2.8-angstrom resolution, J Bacteriol.188, 3391-3401 (2006)26.Boonserm P., Davis P., Ellar D. J. and Li J., Crystal structure of the mosquito-larvicidal toxin Cry4Ba and its biological implications, J Mol Biol, 348, 363-382 (2005)27.Morse R. J., Yamamoto T. and Stroud R. M., Structure of Cry2Aa suggests an unexpected receptor binding epitope, Structure9, 409-417 (2001) 28.Thompson J. D., Higgins D. G. and Gibson T. J., CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Res , 22, 4673-4680 (1994)29.Felsenstein J. Inferring phylogeny, Sinauer Associates, Sunderland, MA (2003)30.Altschul S.F., Madden T.L., Schäffer1 A. A., Zhang J., Zhang Z., Miller W. and Lipman D. J., Gapped BLAST Nucleic Acids Res and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res, 17, 3389-3402 (1997)31.Schwede T.,Kopp J., Guex N. andPeitsch M. C., SWISS-MODEL: an automated protein homology-modeling serve,. Nucl. Acids Res, 31, 3381-3385 (2003). 32.Zhang Y., I-TASSER server for protein 3D structure prediction, BMC bioinformatics, 9, 40-47 (2008)33.Colovos C. and Yeates T. O., Verification of protein structures: Patterns of nonbonded atomic interactions, Protein Science,2,1511-1519 (1993)34.Fiser A., Do R. K. and Sali A., Modeling of loops in protein structures. Protein Science9, 1753-1773(2000)35.Guex N. and Peitsch M. C., SWISS-MODE: and the Swiss-PdbViewer: An environment for comparative protein modeling, Electrophoresis, 18, 2714-2723 (1997) 36.Holm L. and Rosenstrom P. Dali server: conservation mapping in 3D, Nucl Acids Res38, 545-549 (2010)37.Laskowski R. A. MacArthur M. W. Moss D. and Thornton J. M., PROCHECK: a program to check the stereochemical quality of protein structures, J. Appl. Cryst,26, 283-291(1993)38.Wiederstein M. and Sippl M. J., ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins, Nucleic Acids Res , 35, W407-W410 (2007) 39.Ritchie D.W., Evaluation of protein docking predictions using Hex 3.1 in CAPRI rounds 1 and 2. Proteins, Structure, Function, and Bioinformatics52, 98–106 (2003)40.Ramachandraiah G., Chandra N. R., Surolia A. and Vijayan M., Re-refinement using reprocessed data to improve the quality of the structure: a case study involving garlic lectin, Acta Crystallogr D Biol crtstallogr, 58, 414-420 (2002)41.Burton S. L., Ellar D. J., Li J. and Derbyshire D. J., N-acetylgalactosamine on the putative insect receptor aminopeptidase N is recognised by a site on the domain III lectin-like fold of a Bacillus thuringiensis insecticidal toxin, J. Mol. Biol.287, 1011–1022 (1999)42.Hofmann C., Vanderbruggen H., Hofte H., Van Rie J., Jansens S. and Van Mel- laert H., Specificity of Bacillus thuringiensis delta-endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midgets, Proc. Natl. Acad. Sci 85,7844–7848 (1988)43.Jin X., Walker M. A., Felsovalyi K., Vendome J., Bahna F., Mannepalli S., Cosmanescu F., Ahlsen G., Honig B. and Shapiro L., Crystal structures of Drosophila N-cadherin ectodomain regions reveal a widely used class of Ca²+-free interdomain linkers, Proc Natl Acad Sci, U S A 3, 127-134 (2012) 44.Gribskov M., McLachlan A. D. and Eisenberg D., Profile analysis: Detection of distantly related proteins, Proc. Natl. Acad. Sci.84, 4355-4358 (1987) 45.Krogh A., Brown M., Mian I. S., Sjolander K. and Haussler D., Hidden Markov models in computational biology: Applications to protein modeling, J. Mol. Biol.235, 1501- 1531 (1994) 46.Eddy S. R., Hidden Markov models, Curr. Opin. Struct. Biol.6, 361-365 (1996)47.Kashyap S., Singh B.D. and Amla D.V., Homology modeling deduced structure of the Cry1Ab22 toxin, Indian journal of biotechnology,10, 202-206 (2011) 48.Tanje S., Sanam R., Gundla R., Gandhi N.S., Mancera R.L., Boddupally D., Vudem D. R. and Khareedu V. R., Molecular modeling of Bt Cry1Ac (DI-DII)-ASAL (Allium sativum lectin)-fusion protein and its interaction with aminopeptidase N (APN) receptor of Manduca sexta, J. Mol. Graphics Modell, 33, 61-76 (2012)