Correlative effect on the toxicity of three surface‐exposed loops in the receptor‐binding domain of the Bacillus thuringiensis Cry4Ba toxin (original) (raw)
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FEMS Microbiology Letters, 2005
Loop residues in domain II of Bacillus thuringiensis Cry d-endotoxins have been demonstrated to be involved in insecticidal specificity. In this study, selected residues in loops b6-b7 (S 387 SPS 390 ), b8-b9 (S 410 , N 411 , T 413 , T 415 , E 417 and G 418 ) and b10-b11 (D 454 YNS 457 ) in domain II of the Cry4Ba mosquito-larvicidal protein were changed individually to alanine by PCR-based directed mutagenesis. All mutant toxins were expressed in Escherichia coli JM109 cells as 130-kDa protoxins at levels comparable to the wild type. Only E. coli cells that express the P389A, S410A, E417A, Y455A or N456A mutants exhibited a loss in toxicity against Aedes aegypti mosquito larvae of approximately 30% when compared to the wild type. In addition, E. coli cells expressing double mutants, S410A/E417A or Y455A/N456A, at wild-type levels revealed a significantly higher loss in larvicidal activity of approximately 70%. Similar to the wild-type protoxin, both double mutant toxins were structurally stable upon solubilisation and trypsin activation in carbonate buffer, pH 9.0. These results indicate that S 410 and E 417 in the b8-b9 loop, and Y 455 and N 456 in the b10-b11 loop are involved in larvicidal activity of the Cry4Ba toxin.
Journal of Biological Chemistry, 1996
Alanine substitutions of loop 3 residues, 438 SGF-SNS 443 , of CryIAb toxin were constructed to study the functional role of these residues in receptor binding and toxicity to Manduca sexta and Heliothis virescens. Experiments with trypsin and insect gut juice enzyme digestions of mutant toxins showed that these mutations did not produce any gross structural changes to the toxin molecule. Bioassay data showed that mutant G439A (alanine substitution of residue Gly 439) and F440A significantly reduced toxicity toward M. sexta and H. virescens. In contrast, mutants S438A, S441A, N442A, and S443A were similar or only marginally less toxic (2-3 times) to the insects compared to the wild-type toxin. Binding studies with brush border membrane vesicles prepared from M. sexta and H. virescens midgut membranes revealed that the loss of toxicity of mutants G439A and F440A was attributable to substantially reduced initial binding. Consistent with the initial binding, mutants G349A and F440A showed 3.5 times less binding to M. sexta and H. virescens brush border membrane vesicles, although the off-rate of bound toxins was not affected. The role of hydrophobic residue, Phe 440 , is distinctly different from our previous observation that alanine substitution of Phe 371 at loop 2 of CryIAb did not affect initial binding but reduced irreversible association of the toxin to the receptor or membrane toward M.
Febs Letters, 2005
Bacillus thuringiensis subs israelensis produces Cry toxins active against mosquitoes. Receptor binding is a key determinant for specificity of Cry toxins composed of three domains. We found that exposed loop α-8 of Cry11Aa toxin, located in domain II, is an important epitope involved in receptor interaction. Synthetic peptides corresponding to exposed regions in domain II (loop α-8, β-4 and loop 3) competed binding of Cry11Aa to membrane vesicles from Aedes aegypti midgut microvilli. The role of loop α-8 of Cry11A in receptor interaction was demonstrated by phage display and site-directed mutagenesis. We isolated a peptide-displaying phage (P5.tox), that recognizes loop α-8 in Cry11Aa, interferes interaction with the midgut receptor and attenuates toxicity in bioassay. Loop α-8 mutants affected in toxicity and receptor binding were characterized.
Biochemical and Biophysical Research Communications, 2013
We previously demonstrated that co-expression in Escherichia coli of Bacillus thuringiensis (Bt) subsp. israelensis Cry4Ba and Bt subsp. darmstadiensis Cyt2Aa2 shows high synergistic toxicity against target mosquito larvae. Here, further insights into synergistic interactions between these two toxins were revealed through bioactivity restoration of particular inactive Cry4Ba-mutant toxins altered within the receptorbinding domain. Specific mutations at b2-b3 (Y332A) or b4-b5 (F364A) loops, but neither at three other b-hairpin loops (b6-b7, b8-b9 and b10-b11) of Cry4Ba, adversely affect toxicity restoration by Cyt2Aa2. Binding analysis using quartz crystal microbalance verified a decrease in binding of these two bioinactive-mutant toxins (Y332A and F364A) to the immobilized Cyt2Aa2. This suggests that Cry4Ba utilizes these two critical aromatic loop-residues, Tyr 332 and Phe 364 , for synergistic toxicity with its alternative receptor-Cyt2Aa2.
Microbiology, 1996
ry, Information on the molecular determinants of receptor recognition, membrane insertion and toxin pore-formation was sought by making 42 single and multiple substitutions of residues 312-314 (GYY), 367-370 (YRRP) and 438-441 (SGFS) in the Baci//us thuringiensis insecticidal CrylAc G-endotoxin by site-directed mutagenesis. These three regions correspond to three putative surface-exposed loops (loops 1,2 and 3, respectively) in domain II of the 6endotoxin, forming the molecular apex of the structure. All except mutants GFY (loop l), YKRA, SRRA, YRKA (loop 2) and TGFS (loop 3) expressed 6endotoxin protein at wild-type levels which was stable upon activation by Pieris brassicae gut extract or trypsin. Toxicity assays for all the fully stable mutants using Manduca sexta larvae showed that 6312, Y367, R368, R369,5438 and 6439 are important for activity. Wild-type toxin was then labelled in vivo with [35S]methionine and heterologous competition binding assays were carried out for all the mutants using brush border membrane vesicles prepared from Manduca sexta midgut. Most and least conservative mutations of G439 and least conservative substitutions of Y367, R368 and R369 reduced the ability of the toxin to bind competitively. The most conservative mutation, S441T, gave significantly increased binding. These results suggested that these four residues play a role in the initial receptor binding step in the toxin mechanism. As no significant effect on binding affinity was observed in relatively non-toxic mutants in which residues 6312 and 5438 were mutated, we suggest that these residues are involved in the subsequent steps of membrane insertion and pore-formation.
Toxins, 2019
Although the C-terminal domain (DIII) of three-domain Cry insecticidal toxins from Bacillus thuringiensis has been implicated in various biological functions, its exact role still remains to be elucidated. Here, the 21-kDa isolated DIII fragment of the 65-kDa Cry4Ba mosquito-specific toxin was analyzed for its binding characteristics toward lipid-bilayer membranes. When the highly-purified Cry4Ba-DIII protein was structurally verified by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, it revealed the presence of a distinct β-sheet structure, corresponding to its structure embodied in the Cry4Ba crystal structure. Binding analysis via surface plasmon resonance (SPR) spectroscopy revealed that the 21-kDa Cry4Ba-DIII truncate displayed tight binding to immobilized liposome membranes in a two-step manner, exhibiting a dissociation rate constant (k d) comparable to the 65-kDa full-length toxin. Also similar to the Cry4Ba full-length toxin, its isolated DIII truncate was able to anchor a part of its molecule into the immobilized membrane as the SPR signal was still detected after prolonged treatment with proteinase K. However, unlike the full-length active toxin, the DIII truncate was unable to induce membrane permeability of calcein-loaded liposomes or ion-channel formation in planar lipid bilayers. Together, our present data have disclosed a pivotal role of C-terminal DIII in serving as a membrane anchor rather than a pore-forming moiety of the Cry4Ba mosquito-active toxin, highlighting its potential mechanistic contribution to the interaction of the full-length toxin with lipid membranes in mediating toxicity.
Microbiology-sgm, 1996
ry, Information on the molecular determinants of receptor recognition, membrane insertion and toxin pore-formation was sought by making 42 single and multiple substitutions of residues 312-314 (GYY), 367-370 (YRRP) and 438-441 (SGFS) in the Baci//us thuringiensis insecticidal CrylAc G-endotoxin by site-directed mutagenesis. These three regions correspond to three putative surface-exposed loops (loops 1,2 and 3, respectively) in domain II of the 6endotoxin, forming the molecular apex of the structure. All except mutants GFY (loop l), YKRA, SRRA, YRKA (loop 2) and TGFS (loop 3) expressed 6endotoxin protein at wild-type levels which was stable upon activation by Pieris brassicae gut extract or trypsin. Toxicity assays for all the fully stable mutants using Manduca sexta larvae showed that 6312, Y367, R368, R369,5438 and 6439 are important for activity. Wild-type toxin was then labelled in vivo with [35S]methionine and heterologous competition binding assays were carried out for all the mutants using brush border membrane vesicles prepared from Manduca sexta midgut. Most and least conservative mutations of G439 and least conservative substitutions of Y367, R368 and R369 reduced the ability of the toxin to bind competitively. The most conservative mutation, S441T, gave significantly increased binding. These results suggested that these four residues play a role in the initial receptor binding step in the toxin mechanism. As no significant effect on binding affinity was observed in relatively non-toxic mutants in which residues 6312 and 5438 were mutated, we suggest that these residues are involved in the subsequent steps of membrane insertion and pore-formation.
FEBS Letters, 2000
We used site-directed mutagenesis to modify the cally cleaved from an inactive protoxin, to an active toxin Bacillus thuringiensis cry3A gene in amino acid residues 350-form within the insect midgut. The activated toxin binds to 354. Two mutant toxins, A1 (Ra4sA,Yss0F, Y3sIF). and A2 receptors in the midgut and is believed to integrate into the _._ _ (R_sA,AYsso,AYssl), showed significantly improved toxicity lipid bilayer of the brush border membrane. Ion channels are O "" against Tenebrio molitor (yellow mealworm). The mutant toxin formed, causing midgut cells to lose their membrane potential. A1 was also more potent against both Leptinotarsa decemlineata If the rate of damage to the midgut exceeds the rate of repair, (Colorado potato beetle) and Cho,somela scripts (cottonwood lesions form. bacteria invade the hemocele, and death results leaf beetle), while A2 displayed enhanced toxicity only in L. from bacterial septicemia. The fi-endotoxins from B.t. comdecemlineata. Competitive binding assays of L. decemlineata prise a group of over 100 related proteins [5}, which were t5 brush border membrane vesicles (BBMV) revealed that binding previously categorized by insecticidal activity [6] but currently affinities for the AI and A2 mutant toxins were ca. 2.5-fold cq" I-higher than for the wild-type Cry3 toxin. S mar bind ng assays by amino acid similarity [7]. The spectrum of toxicity for each O with C. scripta BBMV revealed a ca. 5-fold lower dissociation toxin is relatively narrow, while the collective activity of this rate for the Al mutant as compared to that of Cry3A. group of pesticidal toxins now spans seven orders of insect © 2000 Federation of European Biochemical Societies. and several other invertebrate groups including nematodes. mites and protozoans [8].