Báo cáo khoa học: Analysis of the region for receptor binding and triggering of oligomerization on Bacillus thuringiensis Cry1Aa toxin potx

Analysis of the region for receptor binding and triggering of oligomerization on Bacillus thuringiensis Cry1Aa toxin Fumiaki Obata, Madoka Kitami, Yukino Inoue, Shogo Atsumi, Yasutaka Yoshizawa and Ryoichi Sato Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Japan Keywords Bacillus thuringiensis; Bombyx mori; BtR175; Cry1Aa; oligomerization Correspondence R Sato, Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan Fax: +81 42 388 7277 Tel: +81 42 388 7277 E-mail: ryoichi@cc.tuat.ac.jp (Received June 2009, revised August 2009, accepted 12 August 2009) doi:10.1111/j.1742-4658.2009.07275.x The determination of the receptor-binding region of Cry toxins produced by Bacillus thuringiensis is expected to facilitate an improvement in their insecticidal ability through protein engineering We analyzed the region on Cry1Aa molecules involved in interactions with the cadherin-like protein receptor BtR175 using cysteine-substituted mutant toxins and several synthetic peptides corresponding to the loops in domain In addition, the region necessary to trigger oligomerization was analyzed using these mutant toxins The mutant toxins were modified by two types of molecule, i.e digested fragments of the Cry1Aa precursor with an average molecular mass of kDa and 5-iodoacetamidofluorescein, which has a molecular mass of 515 kDa We examined whether these modifications interfere with the toxin–BtR175 interaction as a result of steric hindrance 5-Iodoacetamidofluorescein modification of R311C, N376C and G442C revealed steric hindrance effects, indicating that R311 on loop 1, N376 on loop and G442 on loop are on the contact face of the toxin–BtR175 interface when Cry1Aa binds to BtR175 Loop is thought to interact with BtR175 directly, as a peptide corresponding to the N-terminal half of loop 2, (365)LYRRIILG(372), has the potential to bind to BtR175 fragments Meanwhile, mutant toxins with cysteine substitutions in loops and were oligomerized by the binding of digested fragments in the activation process without receptor interaction, and the wild-type toxin formed oligomers by interaction with BtR175 fragments These observations suggest that loops and form both a binding region and a sensor region, which triggers toxin oligomer formation Structured digital abstract l MINT-7259673, MINT-7259722, MINT-7259737, MINT-7259757, MINT-7259774, MINT7259791, MINT-7259808, MINT-7259685, MINT-7259707, MINT-7259830: btr175 (uniprotkb:Q9XY09) binds (MI:0407) to cry1Aa (uniprotkb:P0A366) by surface plasmon resonance (MI:0107) Introduction Bacillus thuringiensis (Bt) is a Gram-positive soil bacterium that produces insecticidal proteins, called Cry toxins, during sporulation Various Bt formulations have been used as pesticides, and genetically modified crops carrying Cry genes have been developed [1,2] However, several problems are associated with the Abbreviations APN, aminopeptidase N; BBMVs, brush border membrane vesicles; Bt, Bacillus thuringiensis; BtR175, 175 kDa cadherin-like protein from Bombyx mori; DEAE, diethylaminoethyl; DF, digested fragments of Cry1Aa precursor; GST, glutathione S-transferase; IAF, 5-iodoacetamidofluorescein FEBS Journal 276 (2009) 5949–5959 ª 2009 The Authors Journal compilation ª 2009 FEBS 5949 Receptor binding ⁄ oligomerization of Cry1Aa mutants F Obata et al practical use of Cry toxins, such as the emergence of resistant insects with continuous implementation and the difficulty of screening new Bt strains for pests to which no identified Cry toxin has sufficient toxicity [3–5] To overcome these problems, attempts have been made to improve Cry toxins artificially [6] The mechanism of insecticidal activity of Cry toxins has not been elucidated fully and several conflicting hypotheses have been proposed [7,8] One hypothesis suggests that Cry toxins form a pore on the target cell membrane, as described below Precursors of Cry toxins are activated as a result of partial digestion by proteases in the gut digestive juice of susceptible larvae [9] Activated Cry1A binds to the cadherin-like protein receptor expressed on the brush border membrane (BBM) of epithelial cells of the midgut [10,11] Cry1A toxins undergo a conformational change with a1-helix cleavage and oligomerization [12,13] Cry1A toxin oligomers bind to GPI-anchored receptors [e.g aminopeptidase N (APN) or alkaline phosphatase (ALP)], followed by pore formation on the cell membrane [14,15] This induces osmotic swelling and bursting of the cell, leading to disruption of the midgut tissue and death of the insect [16] The three-dimensional structure of Cry1A toxins has been revealed by X-ray crystallography [17] Cry1Aa toxin is a 60 kDa protein having three domains: domain consists of a bundle of seven helices (a2 is separated into two helices, a2a and a2b, by a short loop); domain has a b-prism structure composed of antiparallel b-sheets and loops; and domain has a b-sandwich structure composed of two antiparallel b-sheets Domain 2, especially some of its loops, has been shown to play a critical role in receptor binding [18,19] Many analyses of the receptor-binding region have been performed using various Cry toxin mutants For example, mutations of loops 1, and of Cry1Aa have been found to affect significantly the binding activity to BBM vesicles (BBMVs) and the toxicity to Bombyx mori [19–21] Similarly, such loops in Cry1Ab, Cry1Ac and Cry1C are also important in binding to the BBMVs of target insects [20,22,23] Moreover, the binding regions for individual receptor proteins included in BBMVs have been reported For example, the two arginines, 368 and 369, conserved in loop of Cry1Ab and Cry1Ac, are associated with binding to APN [24] Two arginine residues localized in a8 of Cry1Ac have also been indicated to be important in binding to each APN from Manduca sexta and Lymantria dispar [25] In addition, alanine mutations of amino acid residues in loop of Cry1Aa and Cry1Ab reduced binding to cadherin-like protein BtR1 5950 and APN from M sexta and Heliothis virescens [22] However, we cannot exclude the possibility that the loss of binding ability is derived from disruption of the toxin conformation caused by the mutation introduced distant from the receptor-binding site Indeed, mutations in the region distant from the binding site cause unsuspected changes in binding ability in the case of antigen–antibody interactions [26] To determine the binding region directly without amino acid substitutions, loop regions were produced as synthetic peptides and their binding abilities were investigated Synthetic peptides corresponding to loops and inhibited the interaction of Cry1Aa toxins and BtR1 from M sexta, whereas synthetic peptides corresponding to loops a8 and blocked Cry1Ab toxins from binding to BtR1 [27,28] In addition, synthetic peptides corresponding to loop interrupted the binding of Cry1Ac toxins to the H virescens cadherin-like protein, HevCaLP [29] These experimental data suggest that loops a8, and may be associated with receptor binding, but the interaction between Cry1Aa toxins and BtR175 has not been examined to date Recently, we have reported the establishment of a system to improve Cry toxins in vitro using phage display, which is a well-known tool for directed molecular evolution [30] In this system, random mutations are introduced into the toxins and mutants are displayed on phages to prepare a phage library Then, mutant toxins with high affinities to the receptor are screened using high-throughput selection systems, such as panning As the affinity of Cry toxins for receptors is correlated with toxicity [31], some of the mutants with high affinities to the receptor should also show strong insecticidal activity Studies on molecular evolutionary engineering of antibodies have suggested that the induction of mutations in or close to the region interacting with the receptor is effective in generating highaffinity mutant toxins [26] Therefore, the analysis of the receptor-binding region in more detail is needed to determine which region(s) should be mutated In this experiment, we adopted three approaches: (a) the construction of cysteine-substituted mutant toxins and the analysis of their binding activity to the receptor to investigate the role of mutated amino acid residues; (b) the analysis of the influence of the modification of the cysteine residues of mutants with 5-iodoacetamidofluorescein (IAF), which induces steric hindrance; and (c) the analysis of synthetic peptides that can mimic the binding function of Cry1Aa toxin loop regions to the receptor Using these analyses, we can estimate a receptor-interacting region and a region that is in close proximity to the receptor at the toxin–receptor-binding interface FEBS Journal 276 (2009) 5949–5959 ª 2009 The Authors Journal compilation ª 2009 FEBS Receptor binding ⁄ oligomerization of Cry1Aa mutants F Obata et al In addition, we found that a proportion of the cysteine-substituted mutant toxins oligomerized spontaneously during the process of activation by trypsin Oligomerization is considered to be an important step in the insecticidal activity of Cry toxins [32] Several studies using Cry1Ab have indicated that it forms a pre-pore structure before insertion, and various Cry toxin oligomers have been analyzed [13,32–34] For Cry1Aa, the oligomer toxins triggered by BBMVs from M sexta and B mori have been analyzed [35,36], but the type of molecule and the region(s) on the Cry1Aa toxin that is involved in toxin oligomerization remain unclear Therefore, we dissected the mechanism by which Cry1Aa toxins undergo oligomerization using these mutant toxins A Wt – + DTT R281C R311C S373C – + – + – + 60 kDa B 3 a b Results Production and activation of cysteine-substituted mutant toxins and their modification Precursors of cysteine-substituted mutant toxins, R281C, Q293C, R311C, S373C, N376C, G442C and Y445C, were all produced in Escherichia coli as glutathione transferase (GST)-fusion recombinant proteins These toxins were activated by trypsin in the anion exchange column and eluted as purified activated toxins, as reported by Nagamatsu et al [37] The activated mutant toxins were then analyzed by SDSPAGE under nonreducing conditions, and a band slightly larger than the wild-type toxin and an additional smear were observed (Fig 1A) This larger band and smear shifted to the same size as the wildtype toxin on reduction with dithiothreitol (Fig 1A), indicating that certain peptides were bound to the cysteine-substituted mutant toxins via the disulfide bonds of cysteine residues In addition, the molecular masses of the peptides isolated from a mutant toxin treated with dithiothreitol were analyzed using Tricine SDS-PAGE, and the results indicated a broad band spreading from to kDa (data not shown) As described in the Discussion section, these peptides were suggested to be digested fragments (DFs) derived from protoxin, and presumably convenient to use as sources of steric hindrance Therefore, we used these DF-bound toxins, as well as IAF-modified toxins, to analyze the site-specific effects of steric hindrance Conversely, nonmodified mutant toxins were prepared by dithiothreitol reduction of DFbound purified toxins After the reduction and removal of the DFs, IAF modification was performed and checked by western blotting using an anti-fluorescein IgG (Fig 1B) Wt R281C Fig DF and IAF modifications of cysteine-substituted mutant toxins (A) Cysteine-substituted mutant toxins were subjected to nonreducing SDS-PAGE with (+) or without ()) dithiothreitol (DTT) treatment Wt, wild-type toxin (B) Coomassie brilliant blue staining (a) and western blotting using an anti-fluorescein IgG (b) were performed after SDS-PAGE of R281C and the wild-type toxin, which were reduced with dithiothreitol and then modified with IAF IAF modification was specific for the mutant toxins Lane 1, no treatment (DF-bound toxin); lane 2, dithiothreitol reduction (nonmodified toxin); 3, IAF reaction (IAF-modified toxin) Quantitative binding analysis of cysteine-substituted mutant toxins First, we performed binding analyses of nonmodified mutant toxins to BtR175 fragments immobilized on the cuvette of an IAsys optical biosensor Figure shows the overlaid binding curves of the wild-type toxin and all nonmodified mutant toxins The mutant toxins R281C, Q293C and G442C showed almost the same levels of binding to BtR175, whereas Y445C exhibited lower binding activity than the wild-type toxin In contrast, R311C (localized on loop 1), S373C and N376C (both localized on loop 2) showed much higher binding activity than the wild-type toxin Oligomerization of cysteine-substituted mutant toxins Although the affinity of Cry1Aa toxins to BtR175 may be improved by cysteine substitution, the generation of mutants with improved affinity at such high rates seems unlikely Therefore, we postulated that these mutants had oligomerized during preparation If FEBS Journal 276 (2009) 5949–5959 ª 2009 The Authors Journal compilation ª 2009 FEBS 5951 Receptor binding ⁄ oligomerization of Cry1Aa mutants F Obata et al A Temp.(°C) 50 50 30 50 70 100 B N376C 250 Resonance (arc s) 200 R311C 116 97 116 97 S373C 150 200 66 66 Q293C R281C G442C 50 C Wild type Y445C –50 –50 150 350 550 Time (s) 750 – + –+ D 200 oligomerization occurs, the apparent amount of bound toxin increases because several toxin molecules can bind to one receptor First, we characterized the wild-type Cry1Aa toxin oligomer After incubation of the toxin with BBMVs, the band with a molecular mass exceeding 200 kDa was detected with the 60 kDa monomer by western blotting using an anti-Cry1Aa serum (Fig 3A) From the size, we speculated that the protein corresponding to the 200-kDa band had an oligomeric (perhaps tetrameric) structure, formed by the assembly of Cry1Aa toxin monomers The oligomeric structure increased between 15 and h of incubation with BBMVs, and dissociated to the monomer with treatment at 70 °C or above in SDS-PAGE sample buffer (Fig 3A) Furthermore, we showed that oligomerization was induced by the BtR175 fragments used for the binding analysis to an extent similar to BBMVs (Fig 3B) Next, we analyzed the mutant toxin oligomers that showed higher binding to BtR175 than the wild-type toxin (Fig 2) The wild-type toxin and R311C were analyzed before and after incubation with BBMVs For the wild-type toxin, the oligomeric band was observed only after incubation, whereas it was observed even before triggering by BBMVs for the R311C mutant toxin (Fig 3C) This band disappeared almost entirely on of incubation at 95 °C in SDS-PAGE sample buffer, similar to the wild-type oligomeric band (data not shown) These data clearly 200 116 97 Fig Binding analysis of cysteine-substituted mutant toxins to the BtR175 fragment using an IAsys optical sensor Each cysteinesubstituted mutant toxin was added at 150 nM to the cuvette on which the BtR175 fragment was immobilized, and the binding curve was analyzed for 10 After changing the solution to buffer, the dissociation curve was analyzed for For wild-type Cry1Aa, the binding and dissociation curves were analyzed for and min, respectively 5952 BBMV 116 97 66 66 Wt R311C Fig Oligomerization of Cry1Aa wild-type and cysteine-substituted mutant toxins (A) Oligomerization of Cry1Aa wild-type toxin triggered by BBMVs Aliquots of 20 lL of 100 nM of wild-type Cry1Aa were mixed with lg of BBMVs and incubated for 15 or h at 37 °C Samples were then mixed with SDS-PAGE sample buffer and incubated for at various temperatures, as indicated above the figure Each sample was subjected to 7.5% SDS-PAGE and transferred onto nitrocellulose membranes, and the toxins were visualized by western blotting using anti-Cry1Aa serum Lane 1, without BBMVs; lane 2, 15 of incubation with BBMVs; lanes 3–6, h of incubation with BBMVs (B) Oligomerization of Cry1Aa wild-type toxin triggered by BtR175 as well as BBMVs Aliquots of 20 lL of 10 nM Cry1Aa were mixed with 0.5 lg of BBMVs, BtR175 or BSA, and incubated for h at 37 °C Then, the samples were mixed with SDS-PAGE sample buffer and incubated for at 50 °C The oligomeric structure was detected by western blotting as in (A) Lane 1, BBMVs; lane 2, BtR175 fragments; lane 3, BSA (C) Oligomerization of R311C Oligomerization of R311C was analyzed with or without BBMV triggering under the same conditions as in (B) Wt, wild-type toxin (D) Western blotting of each cysteine-substituted mutant toxin after HPLC purification without BBMV triggering using anti-Cry1Aa serum Lane 1, R281C; lane 2, Q293C; lane 3, R311C; lane 4, S373C; lane 5, N376C; lane 6, G442C; lane 7, Y445C show that R311C forms oligomers without any interaction with BBMVs Next, we analyzed all the mutant toxins to determine whether oligomerization had already occurred before interaction with BBMVs, as in R311C (Fig 3D) S373C and N376C mutants demonstrated strong oligomeric bands In contrast, although R281C, G442C and Y445C mutants showed oligomerization, their ratios were quite small We then examined the N-terminal sequence of the oligomeric band using S373C, and obtained the two sequences GIFGP and GPSQW These are sequences from residues 66 and 69, respectively, both of which are located in the loop FEBS Journal 276 (2009) 5949–5959 ª 2009 The Authors Journal compilation ª 2009 FEBS Receptor binding ⁄ oligomerization of Cry1Aa mutants F Obata et al Analysis of the binding ability of synthetic peptides To evaluate the binding capability of the loop regions of Cry1Aa toxins directly, we used eight synthetic peptides corresponding to seven loops (Fig 5A, Table 1): loop a8 [(276)FDGSFRGM(283)], loop [(311) RG(312)], loop [(368)RIILGSGPNNQ(378)], loop [(439)QAAGAVY(445)], loop a8–b2 [(290)NIRQPHLM(297)], loop b4–b5 [(336)PLFGNAGNAAPP (347)] and loop b8–b9 [(404)QRG(406)] The first four of these loops have been reported to be associated with receptor binding Loop was divided into two halves because it is longer than the others The abilities of these eight peptides to bind to BtR175 fragments were analyzed by IAsys, and only P5-1, corresponding to the first half of loop 2, showed a higher binding activity (Fig 5B) Resonance (arc s) Regardless of whether the toxin does or does not oligomerize, modification at cysteine residues can affect the binding affinity of the BtR175 fragment as a result of steric hindrance Therefore, we next analyzed the effects of DF and IAF modifications, which show greater and smaller hindrance, respectively, for the mutant toxins (Fig 4) First, we focused on R281C, Q293C and G442C, all of which showed almost the same binding curve as the wild-type toxin (Fig 2) For R281C, a decrease in binding was seen in the DF-bound form, but not in the IAF-modified form Although even the DF-bound form of Q293C demonstrated high binding ability, G442C showed reduction of binding in both DFbound and IAF-modified forms Next, we analyzed R311C, S373C and N376C, all of which showed increased binding compared with the wild-type toxin (Fig 2) Unexpectedly, increased binding was observed in the DF-bound form of R311C, although the IAFmodified form showed reduced binding capability S373C displayed decreased binding only in the DFbound form and not in the IAF-modified form, similar to R281C, whereas N376C showed decreased binding in both DF-bound and IAF-modified forms, similar to G442C In addition, the binding curve of DF-bound Y445C was the same as that of the nonmodified form, which showed lower affinity (data not shown) Q293C 55 Non–labelled Non–labelled 40 40 IAF 25 25 DF DF 10 10 –5 –50 200 450 700 200 450 700 S373C 110 Non–labelled 270 –5 –50 R311C 420 Resonance (arc s) Binding analysis of DF-bound toxins and IAF-modified toxins R281C 55 Non–labelled 80 DF IAF 50 DF 120 20 –30 –50 IAF 200 450 700 –10 –50 200 450 700 G442C N376C 270 Resonance (arc s) between a-helices 2a and 2b Therefore, at least for S373C, the oligomerized toxin was cleaved at this position, and a-helices and 2a were ablated 40 Non–labelled Non–labelled 195 25 120 DF IAF DF IAF 10 45 –30 –50 –5 200 450 700 Time (s) –50 200 450 700 Time (s) Fig Influence of DF and IAF modifications of cysteine-substituted mutant toxins on binding to BtR175 Nonmodified, DF-bound or IAF-modified toxin was added at a concentration of 150 nM to the cuvette on which BtR175 fragments were immobilized, and the binding curve was analyzed for 10 IAF-modified Q293C was not analyzed because DF-bound toxin showed no difference from nonmodified toxin Discussion DFs bind spontaneously to cysteine-substituted mutant toxins in the activation process We used cysteine-substituted mutant toxins to analyze the receptor-binding region Cysteine was chosen because of the possibility of directed modification by reaction with the thiol group in a protein with no other free thiol groups By substitution of a certain residue for cysteine, we can obtain a mutant toxin with only one cysteine, as activated wild-type Cry1Aa FEBS Journal 276 (2009) 5949–5959 ª 2009 The Authors Journal compilation ª 2009 FEBS 5953 Receptor binding ⁄ oligomerization of Cry1Aa mutants F Obata et al contains no cysteine Mutant toxins formed a broad band slightly larger than that of the wild-type toxin after activation and purification by anion-exchange diethylaminoethyl (DEAE)-HPLC (Fig 1A) In addition, reduction of these toxins with dithiothreitol resulted in the elution of peptides of 1–4 kDa (data not shown) The proteins present in a DEAE column at the time of activation by trypsin were only the precursors of mutant toxins and trypsin Trypsin is a protease that cleaves specifically at the C-terminal end of basic amino acids From the amino acid sequence of Cry1Aa, various types of DFs with cysteine and a molecular mass of 1–4 kDa would be expected to be produced from the C-terminal half of Cry1Aa precursors by tryptic digestion Therefore, peptides bound to cysteine-substituted mutant toxins were inferred to be DFs derived from the C-terminal half of the toxin precursor In addition, we analyzed the N-terminal sequences of R281C and Q293C after HPLC purification, and obtained the sequence of the activated toxin, which begins from amino acid 29 However, the sequences of peptides expected to bind to the cysteinesubstituted mutant toxins were not identified These data suggest that various DFs were bound to the mutant toxins Toxin-binding DFs seem to range in size from to kDa, as observed on SDS-PAGE (Fig 1A, data not shown), suggesting that they can exert wider steric hindrance effects than IAF That is, in comparison with DF, the inhibitory effect of IAF should be restricted to a narrower region A P2 P6 P1 P4 P3 P5-2 P7 P5-1 Resonance (arc s) B 225 P5-1 150 P3 75 –50 P6 P7 P5-2 100 P1 P4 250 400 Time (s) P2 550 The region on Cry1Aa toxins for contact and interaction with BtR175 Fig Binding analysis of synthetic peptides to BtR175 fragments (A) Locations of the synthetic peptides P5-1, which is indicated highest binding property binding to BtR175 fragments in (B), is shown as a dot model (B) Binding analysis of synthetic peptides by IAsys Each synthetic peptide was added to the cuvette at mM, and the binding curve was analyzed until it reached equilibrium Table Sequences of synthetic peptides and corresponding loop regions Synthetic peptide Loop Sequence P1 P2 P3 P4 P5-1 P5-2 P6 P7 a8 a8–b2 b4–b5 2 b8–b9 (276)FDGSFRM(283) (290)NIRQPHLM(297) (309)VHRGFN(314) (338)FGNAGNAAP(346) (365)LYRRIILG(372) (373)SGPNNQEL(380) (401)IYRQRGTV(408) (439)QAAGAVY(445) 5954 From the results of the IAsys binding analysis, the cysteine substitutions at R281, Q293 and G442 did not seem to influence the conformation of Cry1Aa toxins, as the binding abilities of these mutants to BtR175 were no different from that of the wild-type toxin (Fig 2) Three cysteine-substituted mutant toxins, R311C, S373C and N376C, which showed apparent increases on BtR175 binding, were demonstrated to have adopted an oligomeric structure during the activation process (Fig 3D) However, analyses of the contact region or the region in close proximity to BtR175 at the toxin–receptor-binding phase were expected to be implemented without a negative influence of oligomerization by analyzing the effects of modification of cysteine residues Therefore, we examined the regions that influenced the binding ability to BtR175 by DF and IAF modifications N376 located on loop and G442 located on loop were indicated to be in the contact face in the BtR175-binding phase, FEBS Journal 276 (2009) 5949–5959 ª 2009 The Authors Journal compilation ª 2009 FEBS Receptor binding ⁄ oligomerization of Cry1Aa mutants F Obata et al as both DF and IAF modifications showed significant steric hindrance (Fig 4) Unlike these two mutants, the DF modification of R311C increased the apparent binding ability to BtR175 (Fig 4) Although the actual reason is still unknown, bound DFs may have assisted in R311C binding to BtR175 In contrast, IAF modification inhibited the binding of R311C (Fig 4) Both phenomena may indicate that R311 is in close proximity to the BtR175 molecule in the toxin–receptor-binding phase In addition, R281 located on loop a8 and S373 located on loop were shown to be in relatively close proximity to the BtR175 molecule, but not critically in the contact face of the binding phase, because only the DF modification inhibited the interaction (Fig 4) By mapping cysteine-substituted amino acid residues on the Cry1Aa toxin three-dimensional structure, R311, N376 and G442 were located relatively close to each other and R281 and S373 were not far from these residues (Fig 6) Previously, we have reported that the anti-Cry1Aa toxin monoclonal antibody 2A11, which inhibits the binding of Cry1Aa toxin to BtR175, appears to have an epitope in the region consisting of G372, S373, G439, A440, G442, Y445 and T445 [38], which is consistent with the results of the present study As the Y445C mutant loses its binding ability as a result of a cysteine-substituted mutation (Fig 2) and is located in the putative binding region between Cry1Aa toxin and BtR175 (Fig 6), Y445 may have a critical role in binding to BtR175 However, the observation that synthetic peptide P7, which includes Y445, has little affinity to BtR175 (Fig 5) suggests that Y445 substitution for cysteine may alter the conformation of the toxin Moreover, P5-1 showed higher binding ability compared with the other peptides (Fig 5) This observation strongly suggests that a region involved in interactions with BtR175 exists in (365)LYRRIILG(372) of loop In a previous study, an alanine substitution or deletion of 365–372 in loop of Cry1Aa toxins resulted in 1000-fold less toxicity than the wild-type toxin to B mori [19] In addition, alanine or glutamic acid substitution of two arginine residues at positions 368 and 369 of Cry1Ab and Cry1Ac toxins decreased the toxicity to L dispar and M sexta [24] As lysine substitution of these two arginine residues showed no effect on toxicity or receptor-binding ability, a basic amino acid at this site seems to be important [24] Only a few residues are conserved within loop of Cry1Aa, Cry1Ab and Cry1Ac, although two arginine residues are conserved This observation also indicates that the N-terminal half of loop plays a key role in the interaction between Cry1Aa toxins and BtR175 Q293C R281C Loop α8 Loop Loop Loop Y445C G442C R311C N376C S373C Q293C R281C Y445C G442C R311C N376C S373C Fig Mapping of the mutation site Red indicates the amino acid residues that showed inhibition of BtR175 binding by DF binding Yellow indicates the amino acid residues that showed inhibition of BtR175 binding by both DF binding and IAF modification Blue indicates the amino acids that showed inhibition of BtR175 binding without modification Light gray indicates the amino acids that did not inhibit BtR175 binding even with DF binding Top, cartoon model; bottom, surface model Oligomerization of cysteine-substituted mutant toxins without receptor binding Previously, the oligomer of a Cry toxin has been reported to show tolerance to heating at 100 °C [13] However, we could not detect oligomers under such temperature conditions, and thus oligomers were thought to degrade in the SDS-PAGE sample buffer at high temperatures (data not shown) We therefore optimized the conditions to observe the oligomers by SDS-PAGE The oligomer was shown to decompose above 70 °C in the sample buffer (Fig 3A), which was consistent with the results of Ihara and Himeno [36] We analyzed the oligomeric form of the Cry1Aa wildtype toxin using these conditions, and showed that almost all of the toxin molecules existed in monomeric form after HPLC purification, and oligomerization FEBS Journal 276 (2009) 5949–5959 ª 2009 The Authors Journal compilation ª 2009 FEBS 5955 Receptor binding ⁄ oligomerization of Cry1Aa mutants F Obata et al was triggered by cadherin-like protein BtR175 or BBMVs from B mori (Fig 3B) R311C, as well as S373C and N376C, spontaneously oligomerized during the activation process in DEAEHPLC without interaction with BtR175 or BBMVs, in contrast with the wild-type (Fig 3C, D) Cry1Aa toxins contain two helices, a2a and a2b, the C-termini of which are located at residues 63 and 71, respectively [17] Although only a1 was cleaved when Cry1Ab was oligomerized and the N-terminal 60 residues were removed when Cry1Ac was oligomerized [13,39], a1 and a2a were ablated from the N-terminus, resulting in the removal of more than 65 residues when Cry1Aa toxins were oligomerized (this study) ´ Considering the hypothesis of Gomez et al [13], the oligomerization of mutant toxins requires interaction with the receptor or a receptor-like molecule at the time of tryptic digestion in the HPLC column However, no such receptor-like molecule was present in the column for wild-type toxins, as the wild-type Cry1Aa toxin did not oligomerize in this process In addition, if cysteine-substituted mutant toxins were linked to each other via disulfide bonds, the toxins should have formed dimers The oligomers, however, were suggested to be tetramers (Fig 3), indicating a different mechanism of oligomerization In addition, DFs bound to the mutant toxins via thiol in the process of activation (Fig 1A) Therefore, we concluded that DFs mimicked the receptor and triggered the oligomerization of cysteine-substituted mutant toxins Cysteine-substituted mutant toxins may expose helices a1 and a2a of domain on covalent binding to DFs on their cysteine residues, and the two helices may be subsequently ablated by trypsin, followed by the aggregation of toxin monomers Indeed, the triggering of oligomerization by molecules other than the original receptor has been reported previously: for example, the recombinant monoclonal antibody scFV73, which inhibits Cry1Aa toxin binding to BtR1, and Cyt1Aa, which binds to the receptor-binding region of Cry11Aa [13,40] These observations indicate that a conformational change and subsequent oligomerization can be triggered if some proteins bind to the receptor-binding region of Cry toxin Thus, R311, S373 and N376, or loops and of the Cry1Aa toxin, may be located on a receptor-sensing region that promotes oligomerization That is, these amino acid residues or structures may be present in the receptor-binding region Indeed, even IAF-modified toxins R311C and N376C showed decreased binding ability (Fig 4) In contrast, cysteine-substituted mutant toxins of loop a8, loop and other parts, R281C, Q293C, G442C and Y445C, 5956 showed little oligomer formation without receptor interaction (Fig 3D) Application to protein engineering This research has yielded two important findings related to the improvement of Cry toxin activity First, the results of the present study reveal that the region on the binding interface of the Cry1Aa molecule may be responsible for binding Our data suggest that the binding region is located on or around (365)LYRRIILG(372), R311, N376, G442 and Y445 (Figs 2, and 5), and therefore loops 1, and (especially loop 2) are good candidate regions for the introduction of mutations to obtain mutant toxins with a high affinity to the cadherin-like receptor BtR175 of B mori Indeed, improvement of these loops by mutation has been performed for several Cry toxins For example, loop mutants of Cry1Ab and loop mutants of Cry3Aa have higher insecticidal activity than the wildtype [41,42] Moreover, a Cry4Ba loop mutant, which mimics loop of Cry4Aa, showed toxicity for Culex pipiens, which is not susceptible to Cry4Ba but to Cry4Aa, and this mutant showed binding ability to a protein considered to be a receptor [43] Likewise, changing loops and of the lepidopteran-specific Cry1Aa toxin to those of the Cry4 toxin resulted in mosquitocidal toxicity [44] In this way, mutations in loops of the putative binding region have been shown to enable improvement or alteration of the toxicity of Cry toxins Therefore, we expect the application of this knowledge in the phage display system of Cry toxins described in the Introduction To date, a mutant with high affinity to BtR175 has been isolated from a loop random mutant library [30] The other important result of this study is that toxins oligomerize spontaneously without receptor inter´ action Soberon et al [45] have generated a deletion mutant of a1 of Cry1Ab that oligomerizes spontaneously This mutant toxin showed toxicity to Cry1Abresistant Pectinophora gossypiella with deletion of the cadherin receptor, and to BtR1-silenced M sexta [45] These results indicate that a cadherin-like protein is necessary for the oligomerization of toxins, and if the oligomeric structure is constructed spontaneously, resistance by cadherin-like protein deletion can also be overcome Cysteine-substituted mutant toxins in this study also oligomerized spontaneously without any interaction with cadherin-like proteins, and therefore these toxins are expected to be applicable as insecticides or proteins expressed in transgenic plants for the control of insects with cadherin-like protein mutations FEBS Journal 276 (2009) 5949–5959 ª 2009 The Authors Journal compilation ª 2009 FEBS Receptor binding ⁄ oligomerization of Cry1Aa mutants F Obata et al Materials and methods Preparation of Cry1Aa cysteine-substituted mutant toxins and BtR175 fragment Wild-type Cry1Aa protoxin was expressed as described previously [38] Single cysteine mutants were constructed by site-directed mutagenesis using primers designed to substitute R281, Q293, R311, S373, N376, G442 and Y445 for cysteine (R281C-S, 5¢-TTGATGGTAGTTTTTGTGGAAT GGCTCAGA-3¢; R281C-AS, 5¢-TCTGAGCCATTCCACA AAAACTACCATCAA-3¢; Q293C-S, 5¢-ACCAGAATATT AGGTGTCCACATCTTATGGA-3¢; Q293C-AS, 5¢-TCC ATAAGATGTGGACACCTAATATTCTGT-3¢; R311C-S, 5¢-ATACTGATGTGCATTGTGGCTTTAATTATT-3¢; R3 11C-AS, 5¢-AATAATTAAAGCCACAATGCACATCAGT AT-3¢; S373C-S, 5¢-AATTATACTTGGTTGTGGCCCAA ATAATCA-3¢; S373C-AS, 5¢-TGATTATTTGGGCCACA ACCAAGTATAATT-3¢; N376C-S, 5¢-TTGGTTCAGGCC CATGTAATCAGGAACTGT-3¢; N376C-AS, 5¢-ACAGTT CCTGATTACATGGGCCTGAACCAA-3¢; G442C-S, 5¢TGAGCCAAGCAGCTTGTGCAGTTTACACCT-3¢; G44 2C-AS, 5¢-AGGTGTAAACTGCACAAGCTGCTTGGCT CA-3¢; Y445C-S, 5¢-AGCTGGAGCAGTTTGTACCTTGA GAGCTCC-3¢; Y445C-AS, 5¢-GGAGCTCTCAAGGTACA AACTGCTCCAGCT-3¢) The mutated DNAs were cloned into a GST-tagged expression vector, pGEX-4T3 (GE Healthcare, Little Chalfont, UK), and used for the transformation of the BL21 strain of E coli Each mutant toxin was expressed, activated and purified as described previously [38] Briefly, E coli, which produces each toxin, was cultured in MMI broth with ampicillin at 37 °C, and gene expression was induced by isopropyl thio-b-d-galactoside The inclusion body of protoxin was harvested and solubilized, and then protoxin was applied to a DEAE column (Shodex IEC DEAE-825, Showa Denko, Tokyo, Japan) connected to an HPLC system (WatersÔ 600, Milford, MA, USA), and toxin was activated by 0.5 mgỈmL)1 trypsin for h at 37 °C in the column Activated toxin was eluted using a linear gradient of Tris ⁄ HCl buffer, and the purity was checked by SDS-PAGE The concentration of purified toxin was determined by a Coomassie protein assay kit (Pierce, Rockford, IL, USA) using bovine serum albumin as a standard A 27 kDa fragment of Glu1108–Val1464, which contains the toxin-binding region of cadherin-like protein, BtR175 [37], from B mori, was expressed as a GST-fusion protein in E coli and solubilized as described previously [46] The BtR175 fragment was then purified using a MagneGSTÔ Protein Purification System (Promega, Madison, WI, USA) Preparation of BBMV from B mori BBMVs from the midgut of fifth instar larvae of B mori were prepared according to the method described by Wolfersberger et al [47] Amino acid sequence of mutant toxins and DFs Mutant toxins with or without DFs were subjected to 10% SDS-PAGE under nonreducing conditions and transferred onto a poly(vinylidene difluoride) (PerkinElmer Life Sciences, Boston, MA, USA) membrane The N-terminal sequence of each sample was determined by a ProciseÒ cLC protein sequencer (Applied Biosystems, Foster City, CA, USA) Analysis of DF binding and IAF modification of mutant toxins To identify the binding of DFs after purification, cysteine mutant toxins were subjected to SDS-PAGE under reducing and nonreducing conditions The reducing condition was created by the addition of 2-mercaptoetanol at a concentration of 1% to the SDS-PAGE sample buffer DFs were removed from mutant toxins by reduction for 30 at °C with 10 mm dithiothreitol in NaCl ⁄ Pi buffer, and the reduced toxins were dialyzed in NaCl ⁄ Pi buffer at °C IAF modification was performed according to the manufacturer’s instructions Briefly, IAF was added to a final concentration of 10 lm to lm of toxin in NaCl ⁄ Pi buffer with mm EDTA, and the solution was incubated for h at °C with light shielding Extra IAF was removed by dialysis in NaCl ⁄ Pi buffer Modification was checked by western blotting using biotinylated anti-fluorescein IgG and horseradish peroxidase–streptoavidin conjugate Analysis of binding to BtR175 fragment by IAsys optical sensor Binding analysis using IAsys was conducted according to the method of Atsumi et al [38] Briefly, a 150 nm mutant toxin solution was added to the cuvette on which BtR175 fragment had been immobilized, and the binding curve was recorded for 10 Then, the toxin solution was removed by altering the solution to NaCl ⁄ Pi, and the dissociation curve was analyzed for The cuvette was regenerated after treatment with 20 mm HCl, and the association and dissociation of each mutant toxin were analyzed sequentially For synthetic peptides, mm of solution was added to the cuvette and the binding curve was analyzed until the reaction reached equilibrium Oligomerization of Cry1Aa toxin and detection of oligomerized toxin For the construction of oligomer, 20 lL of a 10 or 100 nm toxin solution were incubated for h at 37 °C with 0.5 or lg BBMV or BtR175 fragment, and the sample was subjected to western blotting As the oligomeric structure is unstable at high temperature in SDS-PAGE sample buffer, each sample was incubated in the buffer for at 50 °C Oligomer was detected using anti-Cry1Aa toxin serum FEBS Journal 276 (2009) 5949–5959 ª 2009 The Authors Journal compilation ª 2009 FEBS 5957 Receptor binding ⁄ oligomerization of Cry1Aa mutants F Obata et al Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (B) (21310051) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan References 15 Crickmore N (2006) Beyond the spore – past and future developments of Bacillus thuringiensis as a biopesticide J Appl Microbiol 101, 616–619 Romeis J, Meissle M & Bigler F (2006) Transgenic crops expressing Bacillus thuringiensis toxins and biological control Nat Biotechnol 24, 63–71 Griffitts JS & Aroian RV (2005) Many roads to resistance: how invertebrates adapt to Bt toxins BioEssays 27, 614–624 ´ Tabashnik BE, Gassmann AJ, Crowder DW & Carriere Y (2008) Insect resistance to Bt crops: evidence versus theory Nat Biotechnol 26, 199–202 ´ Bravo A & Soberon M (2008) How to cope with insect resistance to Bt toxins? Trends Biotechnol 26, 573–579 ´ Pardo-Lopez L, Munoz-Garay C, Porta H, Rodrı´ guez˜ ´ ´ Almazan C, Soberon M & Bravo A (2009) Strategies to improve the insecticidal activity of Cry toxins from Bacillus thuringiensis Peptides 30, 589–595 ´ Soberon M, Gill SS & Bravo A (2009) Signaling versus punching hole: how Bacillus thuringiensis toxins kill insect midgut cells? Cell Mol Life Sci 66, 1337–1349 Pigott CR & Ellar DJ (2007) Role of receptors in Bacillus thuringiensis crystal toxin activity Microbiol Mol Biol Rev 71, 255–281 Aronson AI, Wu D & Zhang C (1995) Mutagenesis of specificity and toxicity regions of a Bacillus thuringiensis protoxin gene J Biol Chem 177, 4059–4065 10 Hofmann C, Luthy P, Hutter R & Pliska V (1988) ă ă Binding of the d-endotoxin from Bacillus thuringiensis to brush-border membrane vesicles of the cabbage butterfly (Pieris brassicae) Eur J Biochem 173, 85–91 11 Keeton TP, Francis BR, Maaty WS & Bulla LA Jr (1998) Effects of midgut-protein-preparative and ligand binding procedures on the toxin binding characteristics of BT-R1, a common high-affinity receptor in Manduca sexta for Cry1A Bacillus thuringiensis toxins Appl Environ Microbiol 64, 2158–2165 12 Gazit E, La Rocca P, Sansom MS & Shai Y (1998) The structure and organization within the membrane of the helices composing the pore-forming domain of Bacillus thuringiensis d-endotoxin are consistent with an ‘umbrella-like’ structure of the pore Proc Natl Acad Sci USA 95, 12289–12294 ´ ´ ´ 13 Gomez I, Sanchez J, Miranda R, Bravo A & Soberon M (2002) Cadherin-like receptor binding facilitates pro- 5958 14 16 17 18 19 20 21 22 23 24 25 teolytic cleavage of helix alpha-1 in domain I and oligomer pre-pore formation of Bacillus thuringiensis Cry1Ab toxin FEBS Lett 513, 242–246 Schwartz JL, Lu YJ, Sohnlein P, Brousseau R, Laprade R, Masson L & Adang MJ (1997) Ion channels formed in planar lipid bilayers by Bacillus thuringiensis toxins in the presence of Manduca sexta midgut receptors FEBS Lett 412, 270–276 ´ ´ Bravo A, Gomez I, Conde J, Munoz-Garay C, Sanchez ˜ ´ J, Miranda R, Zhuang M, Gill SS & Soberon M (2004) Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains Biochim Biophys Acta 1667, 38–46 Knowles BH & Ellar DJ (1987) Colloid-osmotic lysis is a general feature of the mechanisms of action of Bacillus thuringiensis d-endotoxins with different insect specificities Biochim Biophys Acta 924, 509–518 Grochulski P, Masson L, Borisova S, Pusztai-Carey M, Schwartz JL, Brousseau R & Cygler M (1995) Bacillus thuringiensis CryIA(a) insecticidal toxin: crystal structure and channel formation J Mol Biol 254, 447–464 Ge AZ, Rivers D, Milne R & Dean DH (1991) Functional domains of Bacillus thuringiensis insecticidal crystal proteins J Biol Chem 266, 17954–17958 Lu H, Rajamohan F & Dean DF (1994) Identification of amino acid residues of Bacillus thuringiensis d-endotoxin CryIAa associated with membrane binding and toxicity to Bombyx mori J Bacteriol 176, 5554–5559 Rajamohan F, Hussain SRA, Cotrill JA, Gould F & Dean DH (1996) Mutations at domain II, loop 3, of Bacillus thuringiensis CryIAa and CryIAb d-endotoxins suggest loop is involved in initial binding to lepidopteran midguts J Biol Chem 271, 25220–25226 Rajamohan F, Lee MK & Dean DH (1998) Bacillus thuringiensis insecticidal proteins: molecular mode of action Prog Nucleic Acid Res Mol Biol 60, 1–27 Rajamohan F, Cotrill JA, Gould F & Dean DH (1996) Role of domain II, loop residues of Bacillus thuringiensis CryIAb d-endotoxin in reversible and irreversible binding to Manduca sexta and Heliothis virescens J Biol Chem 271, 2390–2397 Abdul-Rauf M & Ellar DJ (1999) Mutations of loop and loop residues in domain II of Bacillus thuringiensis Cry1C delta-endotoxin affect insecticidal specificity and initial binding to Spodoptera littoralis and Aedes aegypti midgut membranes Curr Microbiol 39, 94–98 Lee MK, Rajamohan F, Jenkins JL, Curtiss AS & Dean DH (2000) Role of two arginine residues in domain II, loop of Cry1Ab and Cry1Ac Bacillus thuringiensis delta-endotoxin in toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase N Mol Microbiol 38, 289–298 Lee MK, Jenkins JL, You TH, Curtiss A, Son JJ, Adang MJ & Dean DH (2001) Mutations at the argi- FEBS Journal 276 (2009) 5949–5959 ª 2009 The Authors Journal compilation ª 2009 FEBS Receptor binding ⁄ oligomerization of Cry1Aa mutants F Obata et al 26 27 28 29 30 31 32 33 34 35 nine residues in alpha8 loop of Bacillus thuringiensis delta-endotoxin Cry1Ac affect toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase N FEBS Lett 497, 108–112 Hawkins RE, Russell SJ, Baier M & Winter G (1993) The contribution of contact and non-contact residues of antibody in the affinity of binding to antigen The interaction of mutant D1.3 antibodies with lysozyme J Mol Biol 234, 958–964 Gomez I, Miranda-Rios J, Rudino-Pinera E, Oltean ˜ ˜ ´ DI, Gill SS, Bravo A & Soberon M (2002) Hydropathic complementarity determines interaction of epitope (869)HITDTNNK(876) in Manduca sexta Bt-R(1) receptor with loop of domain II of Bacillus thuringiensis Cry1A toxins J Biol Chem 277, 30137– 30143 ´ ´ Gomez I, Dean DH, Bravo A & Soberon M (2003) Molecular basis for Bacillus thuringiensis Cry1Ab toxin specificity: two structural determinants in the Manduca sexta Bt-R1 receptor interact with loops alpha-8 and in domain II of Cy1Ab toxin Biochemistry 42, 10482– 10489.r Xie R, Zhuang M, Ross LS, Gomez I, Oltean DI, Bravo A, Soberon M & Gill SS (2005) Single amino acid mutations in the cadherin receptor from Heliothis virescens affect its toxin binding ability to Cry1A toxins J Biol Chem 280, 8416–8425 Ishikawa H, Hoshino Y, Motoki Y, Kawahara T, Kitajima M, Kitami M, Watanabe A, Bravo A, Soberon M, Honda A et al (2007) A system for the directed evolution of the insecticidal protein from Bacillus thuringiensis Mol Biotechnol 36, 90–101 Van Rie J, Jansens S, Hofte H, Degheele D & Van ă Mellaert H (1990) Receptors on the brush border membrane of the insect midgut as determinants of the specificity of Bacillus thuringiensis delta-endotoxins Appl Environ Microbiol 56, 1378–1385 ´ ´ ´ Jimenez-Juarez N, Munoz-Garay C, Gomez I, Saab˜ ´ Rincon G, Damian-Almazo JY, Gill SS, Soberon M & Bravo A (2007) Bacillus thuringiensis Cry1Ab mutants affecting oligomer formation are non-toxic to Manduca sexta larvae J Biol Chem 282, 21222–21229 ´ ´ Rausell C, Pardo-Lopez L, Sanchez J, Munoz-Garay C, ˜ ´ Morera C, Soberon M & Bravo A (2004) Unfolding events in the water-soluble monomeric Cry1Ab toxin during transition to oligomeric pre-pore and membraneinserted pore channel J Biol Chem 279, 55168–55175 ´ ´ ´ Pardo-Lopez L, Gomez I, Munoz-Garay C, Jimenez˜ ´ Juarez N, Soberon M & Bravo A (2006) Structural and functional analysis of the pre-pore and membraneinserted pore of Cry1Ab toxin J Invertebr Pathol 92, 172–177 ´ Munoz-Garay C, Sanchez J, Darszon A, de Maagd ˜ ´ RA, Bakker P, Soberon M & Bravo A (2006) Permeability changes of Manduca sexta midgut brush border 36 37 38 39 40 41 42 43 44 45 46 47 membranes induced by oligomeric structures of different cry toxins J Membr Biol 212, 61–68 Ihara H & Himeno M (2008) Study of the irreversible binding of Bacillus thuringiensis Cry1Aa to brush border membrane vesicles from Bombyx mori midgut J Invertebr Pathol 98, 177–183 Nagamatsu Y, Koike T, Sasaki K, Yoshimoto A & Furukawa Y (1999) The cadherin-like protein is essential to specificity determination and cytotoxic action of the Bacillus thuringiensis insecticidal CryIAa toxin FEBS Lett 460, 385–390 Atsumi S, Inoue Y, Ishizaka T, Mizuno E, Yoshizawa Y, Kitami M & Sato R (2008) Location of the Bombyx mori 175 kDa cadherin-like protein-binding site on Bacillus thuringiensis Cry1Aa toxin FEBS J 275, 4913– 4926 Aronson AI, Geng C & Wu L (1999) Aggregation of Bacillus thuringiensis Cry1A toxins upon binding to target insect larval midgut vesicles Appl Environ Microbiol 65, 2503–2507 ´ ´ Perez C, Munoz-Garay C, Portugal LC, Sanchez J, Gill ˜ ´ SS, Soberon M & Bravo A (2007) Bacillus thuringiensis ssp israelensis Cyt1Aa enhances activity of Cry11Aa toxin by facilitating the formation of a pre-pore oligomeric structure Cell Microbiol 9, 2931–2937 Rajamohan F, Alzate O, Cotrill JA, Curtiss A & Dean DH (1996) Protein engineering of Bacillus thuringiensis d-endotoxin: mutations at domain II of Cry1Ab enhance receptor affinity and toxicity towards gypsy moth larvae Proc Natl Acad Sci USA 93, 14338–14343 Wu SJ, Koller CN, Miller DL, Bauer LS & Dean DH (2000) Enhanced toxicity of Bacillus thuringiensis Cry3A delta-endotoxin in coleopterans by mutagenesis in a receptor binding loop FEBS Lett 473, 227–232 Abdullah MA, Alzate O, Mohammad M, McNall RJ, Adang MJ & Dean DH (2003) Introduction of Culex toxicity into Bacillus thuringiensis Cry4Ba by protein engineering Appl Environ Microbiol 69, 5343–5353 Liu XS & Dean DH (2006) Redesigning Bacillus thuringiensis Cry1Aa toxin into a mosquito toxin Protein Eng Des Sel 19, 107–111 ´ ´ ´ ´ Soberon M, Pardo-Lopez L, Lopez I, Gomez I, Tabashnik BE & Bravo A (2008) Engineering modified Bt toxins to counter insect resistance Science 318, 1561–1562 Hara H, Atsumi S, Yaoi K, Nakanishi K, Higurashi S, Miura N, Tabunoki H & Sato R (2003) A cadherin-like protein functions as a receptor for Bacillus thuringiensis Cry1Aa and Cry1Ac toxins on midgut epithelial cells of Bombyx mori larvae FEBS Lett 538, 29–34 Wolfersberger MG, Luthy P, Mauer A, Parenti P, Sacchi V, Giordana B & Hanozet G (1987) Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (Pieris brassicae) Comp Biochem Physiol 86, 301–308 FEBS Journal 276 (2009) 5949–5959 ª 2009 The Authors Journal compilation ª 2009 FEBS 5959 ... function of Cry1Aa toxin loop regions to the receptor Using these analyses, we can estimate a receptor- interacting region and a region that is in close proximity to the receptor at the toxin? ? ?receptor- binding. .. to the receptor- binding region of Cry toxin Thus, R311, S373 and N376, or loops and of the Cry1Aa toxin, may be located on a receptor- sensing region that promotes oligomerization That is, these... From the results of the IAsys binding analysis, the cysteine substitutions at R281, Q293 and G442 did not seem to influence the conformation of Cry1Aa toxins, as the binding abilities of these