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Membrane Insertion of the Bacillus thuringiensis Cry1Ab Toxin: Single Mutation in Domain II Block Partitioning of the Toxin into the Brush Border Membrane †
Abstract
The umbrella and penknife models hypothesize that insecticidal Bacillus thuringiensis Cry toxins partition into the apical membrane of the insect midgut by insertion of only two alpha-helices from domain I of the protein, alpha-helices 4 and 5 in the case of the umbrella model and alpha-helices 5 and 6 in the case of the penknife model. Neither model envisages membrane partitioning by domains II and III. In this study, we present data suggesting that mutations in the domain II residue, F371, affect insertion of the whole toxin into Manduca sexta brush border membrane vesicles (BBMVs). Using steady state fluorescence measurements combined with a proteinase K protection assay, we show that mutants of F371 have lost their ability to insert into the BBMV, even though binding to cadherin is almost unaffected. The study also identifies a difference in partitioning of toxins into artificial lipid vesicles (SUVs) as opposed to native BBMVs. While the F371 mutations block insertion of domains I and II into BBMVs, they only block domain II insertion into SUVs. Bioassay and voltage clamping of midguts also confirm the fluorescence data that the noninserting mutants are nontoxic. Our study leads us to propose that, in contrast to previous models of individual free helices inserting into the membrane, the toxin enters into the membrane as a whole molecule or oligomers of the molecule, wherein the domain II residue F371 has a vital role to play in membrane insertion.
... in 1X TBST. The membrane was treated with the anti-rabbit-HRP (Nair, Liu, & Dean, 2008) secondary (BioRad) for 45 minutes with a 1:10000 dilution and briefly rinsed. The membrane was rinsed 4 times for 8 min. ...
... TBST. The membrane was treated with the anti-rabbit-HRP secondary antibody (BioRad) for 45 minutes with a 1:10000 dilution and briefly rinsed (Nair, et al., 2008). The membrane was rinsed 4 times for 8 min. ...
... Culex pipiens and Aedes aegypti eggs were hatched and reared according to specifications previously outlined (Nair, et al., 2008 Koi, fine particles were retained and larvae were fed 60mg on day two. Larvae were fed 120 to 240mg of ground Nishi Koi on day four and above until pupation of all larvae. ...
... For mutations other than that to Trp, we see further decrease in insertion rate based on ion channel-forming abilities like voltage clamp studies and binding studies to BBMV (34). F371C was the most affected in these studies, and using the thiol probe acrylodan, it was shown that this mutation was completely ineffective in partitioning into brush border membranes (35). Labeling Cry1A Mutants with Fluorophore-Purified cysteine mutants were labeled with acrylodan or IAEDANS and purified off free labels using gel filtration. ...
... The extent of blue shift was not the same before and after proteinase K treatment as indicated in Fig. 4. All these mutants were accompanied by an increase in the intensity of acrylodan fluorescence as seen in case of the representative spectra (Fig. 5, A-C). The only exceptions to this were Cry1Ab L40C, which showed decrease in its fluorescence emission (Fig. 5D), and Cry1Ab F371C, which block the protein from partitioning into the membrane (35). ...
... Slopes for the linear region of the drop in the I sc were calculated ( Table 2 and Table 3). The voltage clamp response for Cry1Ab V171C and F371C are already reported earlier (35). The rate of ion transport was measured as the slope of the linear region of the drop in short circuit current for each of the mutations. ...
A critical step in understanding the mode of action of insecticidal crystal toxins from Bacillus thuringiensis is their partitioning into membranes and, in particular, the insertion of the toxin into insect brush border membranes. The
Umbrella and Penknife models predict that only α-helix 5 of domain I along with adjacent helices α-4 or α-6 insert into the
brush border membranes because of their hydrophobic nature. By employing fluorescent-labeled cysteine mutations, we observe
that all three domains of the toxin insert into the insect membrane. Using proteinase K protection assays, steady state fluorescence
quenching measurements, and blue shift analysis of acrylodan-labeled cysteine mutants, we show that regions beyond those proposed
by the two models insert into the membrane. Based on our studies, the only extended region that does not partition into the
membrane is that of α-helix 1. Bioassays and voltage clamping studies show that all mutations examined, except certain domain
II mutations in loop 2 (e.g. F371C and G374C), which disrupt membrane partitioning, retain their ability to form ion channels and toxicity in Manduca sexta larvae. This study confirms our earlier hypothesis that insertion of crystal toxin does not occur as separate helices alone,
but virtually the entire molecule inserts as one or more units of the whole molecule.
... Cry toxins have suggested that the protein could insert into the midgut membrane as a single molecule, opening the possibility for a third model for the topology of the toxin in the membrane (Aronson et al., 1999; Aronson, 2000; Loseva et al., 2001; Alzate et al., 2006; Nair et al., 2008; Alzate et al., 2009). In this last model, virtually the whole toxin, when associated with BBMV, is protected from proteinase K, with the exception that (in Cry1A toxins) α-helix 1 is cleaved off of the inserted form. ...
... Dado que la toxina completa de Cry1Aa y casi todo de los dominios II y III de L371K están protegidos de proteasas en presencia de VMBC del insecto, este estudio sugiere que la inserción de la toxina en la membrana involucra los tres dominios. with wild type and the mutant protein, have led us to propose that besides domain I, domains II and III have also the ability to penetrate the membrane in support of our previous findings ( Alzate et al., 2006; Nair et al., 2008; Alzate et al., 2009). ...
... A second possibility is that the β-sheets of domain II and III may form oligomers after binding to the receptor, thus protecting the whole toxin from degradation. In this work we support a model for the membrane bound state of the δ-endotoxins, in which the whole toxin inserts into the membrane, in agreement with previous results (Aronson et al., 1999; Aronson, 2000; Loseva et al., 2001; Nair et al., 2008; Alzate et al., 2007; Alzate et al., 2009) that suggests new interpretations for the mechanism of action of these important biopesticides. ...
The surface exposed Leucine 371 on loop 2 of domain II, in Cry1Aa toxin, was mutated to Lysine to generate the trypsin-sensitive mutant, L371K. Upon trypsin digestion L371K is cleaved into approximately 37 and 26 kDa fragments. These are separable on SDS-PAGE, but remain as a single molecule of 65 kDa upon purification by liquid chromatography. The larger fragment is domain I and a portion of domain II (amino acid residues 1 to 371). The smaller 26-kDa polypeptide is the remainder of domain II and domain III (amino acids 372 to 609). When the mutant toxin was treated with high dose of M. sexta gut juice both fragments were degraded. However, when incubated with M. sexta BBMV, the 26 kDa fragment (domains II and III) was preferentially protected from gut juice proteases. As previously reported, wild type Cry1Aa toxin was also protected against degradation by gut juice proteases when incubated with M. sexta BBMV. On the contrary, when mouse BBMV was added to the reaction mixture neither Cry1Aa nor L371K toxins showed resistance to M. sexta gut juice proteases and were degraded. Since the whole Cry1Aa toxin and most of the domain II and domain III of L371K are protected from proteases in the presence of BBMV of the target insect, we suggest that the insertion of the toxin into the membrane is complex and involves all three domains.
... This domain is involved in insertion of protein into the insect's midgut membrane, producing ion pores or ion channels that result in death of the insect (reviewed in reference 12). To probe the active conformation of the Cry1A ␦-endotoxins in the insect's midgut membrane, we created a series of mutant proteins that have been analyzed by biophysical techniques, including electron paramagnetic resonance (2), membrane partitioning assays (1)(2)(3)10), and fluorescence spectroscopy (10). The single mutation of valine 171, located in ␣-helix 5 (Fig. 1A), to cysteine (V171C) resulted in a toxin with a 25-fold increase in toxicity against Lymantria dispar. ...
... This domain is involved in insertion of protein into the insect's midgut membrane, producing ion pores or ion channels that result in death of the insect (reviewed in reference 12). To probe the active conformation of the Cry1A ␦-endotoxins in the insect's midgut membrane, we created a series of mutant proteins that have been analyzed by biophysical techniques, including electron paramagnetic resonance (2), membrane partitioning assays (1)(2)(3)10), and fluorescence spectroscopy (10). The single mutation of valine 171, located in ␣-helix 5 (Fig. 1A), to cysteine (V171C) resulted in a toxin with a 25-fold increase in toxicity against Lymantria dispar. ...
... We used UV-visible spectroscopy (UV/VIS) and circular dichroism (CD) spectroscopy to determine whether the increased toxicity resulted from structural alterations. As previously reported (10), CD revealed that the secondary structures of the mutant and the wild-type toxins are virtually identical. We then studied whether the folding and/or unfolding rate of the toxins showed any differences that could account for the observed changes in toxicity. ...
The Cry1Ab δ-endotoxin V171C mutant protein exhibits a 25-fold increase in toxicity against Lymantria dispar, which correlates with a faster rate of partitioning into the midgut membrane and slightly decreased protein stability. This
is an insect-specific mechanism; similar results were not observed in Manduca sexta, another Cry1Ab δ-endotoxin-susceptible insect.
... The removal of helix α-1 results in the formation of oligomers that are membrane insertion competent ). The binding of Cry toxins to the cadherin-like receptors have been shown to involve specific interactions of the variable loop regions in domain II and III with cadherin epitopes (Nair et al. 2008;Chen et al. 2009;Pacheco et al. 2009a;Soberón et al. 2009). ...
... The oligomerised activated toxin that is bound to membrane receptors then inserts the central hydrophobic helix α-4 and 5 (Nair et al. 2008) into the apical membrane of midgut cells causing osmotic shock, bursting of the midgut cells and finally ending in the insect death (Knowles and Ellar 1987;Haider and Ellar 1989;Grochulski et al. 1995;Schnepf et al. 1998;Bravo et al. 2004;Rausell et al. 2004). The pore formation model as proposed by Bravo et al. (2004) for Cry1A toxins is presented in Fig. 2.3. ...
Bacillus thuringiensis (Bt) and its insecticidal toxins have been used in agronomical pest control for decades. The mechanism of action of Bt toxins on insect pest involves specific molecular interactions which makes Bt a popular choice for pest control. The specificity of action of Bt toxins reduces the concern of adverse effects on non-target species, a concern which remains with chemical insecticides. Different strains of Bt are known to express different classes of toxins which in turn target different insects. Bt and its toxins can be formulated into powder or liquid sprays or expressed in transgenic plants. To maximize the effect of Bt toxins, multiple toxins are often combined when making Bt formulations or expressed in transgenic plants. Though Bt is a very effective biological control agent, there are concerns over the development of resistance by insect species and also the narrow spectrum of activity of individual toxins. To address these concerns, new strains of Bt expressing novel toxins are actively sought and existing toxins are genetically modified for improved activity. © 2012 Springer Science+Business Media B.V. All rights reserved.
... Western blot analysis against Cry2A was performed using previously published protocols (Nair et al., 2008). ...
... A decline in toxicity to this magnitude may infer that receptor binding event was affected or proteolytic degradation in the gut lumen. Alternatively, loss of toxicity may be attributed to the disruption of the membrane insertion event and should be considered (Nair et al., 2008). ...
Cry2Aa exhibits dual activity to Lepidoptera and Diptera. Cry2Ab differs in amino acid sequence from Cry2Aa by 13% and has shown significant lepidopteran activity, but no mosquitocidal activity. Previous studies implicate 23 Cry2Aa specificity-conferring residues of domain II, which differ in Cry2Ab. Nine residues are putatively involved in conferring Cry2Aa dipteran specificity. To explore Cry2Ab dipteran toxicity, site-directed mutagenesis was employed to exchange Cry2Ab residues with Cry2Aa D (dipteran) block residues. Cry2Ab wild type demonstrated high toxicity (LC(50) of 540 ng mL(-1)) to Anopheles gambiae, but not to Aedes or Culex, within a 24-h time period. Cry2Ab should be reclassified as a dual active Cry toxin. Cry2Ab mutagenesis revealed critical residues for Cry2Ab protein function, as well as enhanced activity against the malarial mosquito, An. gambiae.
... In addition, a Cry1Ab domain II loop 2 mutant (Cry1AbF371A) was affected in oligomer binding to APN1 and toxicity (Arenas et al., 2010). This Cry1AbF371A mutant was shown to be affected in insertion into M. sexta BBMV (Nair et al., 2008), suggesting that binding of Cry1Ab oligomers to APN1 or ALP is involved in facilitating oligomer membrane insertion (Arenas et al., 2010) (Fig. 2). A summary figure describing the sequential binding model is shown in Fig. 2. Monomeric Cry toxins preferentially bind first to GPI-anchored receptors thus directing the activated toxin to the membrane and facilitating the interaction of monomeric Cry proteins with the less abundant cadherin or ABC transporters receptors. ...
... As a control in our experiments, we analysed the binding of the non-toxic Cry1Ab F371A mutant that is affected in its irreversible binding to BBMVs [28]. It was reported that the loss of irreversible binding of this mutant is due to a lack of toxin insertion into the membrane [28,61]. It was also shown that the monomeric structure of this mutant was able to bind APN and ALP with a similar affinity as did the wild-type toxin. ...
Cry-proteins from Bacillus thuringiensis are insecticidal pore forming toxins (PFT). Here we show that two distinct functional pre-pores of Cry1Ab are formed after binding of the protoxin or the protease-activated toxin to the cadherin-receptor, but before membrane-insertion. Both pre-pores are active inducing pore formation, although with different characteristics, and contribute to the insecticidal activity. We also analyzed the oligomerization of the mutant Cry1AbMod protein. This mutant kills different insect populations resistant to Cry toxins, but lost potency against susceptible insects. We found that the Cry1AbMod-protoxin efficiently induces oligomerization, and not the activated Cry1AbMod-toxin, explaining the loss of potency of Cry1AbMod against susceptible insects. These data are relevant for the future control of insects resistant to Cry proteins. Our data support the pore formation model involving sequential interaction with different midgut proteins, leading to pore formation in the target-membrane. We propose that not only different insect-targets could have different receptors, but also different midgut proteases that would influence the rate of protoxin/toxin activation. It is possible that the two pre-pore structures could have been selected in evolution, since they have differential roles in toxicity against selected targets, increasing their range of action. These data provide a functional role to the protoxin-fragment of Cry-PFT that was not previously understood. Most PFT produced by other bacteria are secreted as protoxins that require activation before oligomerization, to finally form a pore. Thus different pre-pores could be also part of general mechanism of action of other PFT.
... It is conceivable that residues in domain II are not always involved in receptor binding. For example, the Cry1Ab F371 loop mutant lost its ion channel activity instead of binding activity suggesting a role for this residue in membrane insertion [54,55]. ...
Using a Cry11Ba toxin model, predicted loops in domain II were analyzed for their role in receptor binding and toxicity. Peptides corresponding to loops alpha8, 1 and 3, but not loop 2, competed with toxin binding to Aedes midgut membranes. Mutagenesis data reveal loops alpha8, 1 and 3 are involved in toxicity. Loops 1 and 3 are of greater significance in toxicity to Aedes and Culex larvae than to Anopheles. Cry11Ba binds the apical membrane of larval caecae and posterior midgut, and binding can be competed by loop 1 but not by loop 2 peptides. Cry11Ba binds the same regions to which anti-cadherin antibody binds, and this antibody competes with Cry11Ba binding suggesting a possible role of cadherin in toxication.
... In agreement with these findings, ␣4 was shown to line the lumen of the pores (42). On the other hand, convincing evidence supporting previous suggestions that most of the toxin molecule may become imbedded in the membrane (3,39,60) has recently been reported (44,45). ...
Pore formation in the apical membrane of the midgut epithelial cells of susceptible insects constitutes a key step in the
mode of action of Bacillus thuringiensis insecticidal toxins. In order to study the mechanism of toxin insertion into the membrane, at least one residue in each of
the pore-forming-domain (domain I) interhelical loops of Cry1Aa was replaced individually by cysteine, an amino acid which
is normally absent from the activated Cry1Aa toxin, using site-directed mutagenesis. The toxicity of most mutants to Manduca sexta neonate larvae was comparable to that of Cry1Aa. The ability of each of the activated mutant toxins to permeabilize M. sexta midgut brush border membrane vesicles was examined with an osmotic swelling assay. Following a 1-h preincubation, all mutants
except the V150C mutant were able to form pores at pH 7.5, although the W182C mutant had a weaker activity than the other
toxins. Increasing the pH to 10.5, a procedure which introduces a negative charge on the thiol group of the cysteine residues,
caused a significant reduction in the pore-forming abilities of most mutants without affecting those of Cry1Aa or the I88C,
T122C, Y153C, or S252C mutant. The rate of pore formation was significantly lower for the F50C, Q151C, Y153C, W182C, and S252C
mutants than for Cry1Aa at pH 7.5. At the higher pH, all mutants formed pores significantly more slowly than Cry1Aa, except
the I88C mutant, which formed pores significantly faster, and the T122C mutant. These results indicate that domain I interhelical
loop residues play an important role in the conformational changes leading to toxin insertion and pore formation.
The crystal protein Cry5B, a pore-forming protein produced by the soil bacterium Bacillus thuringiensis, has been demonstrated to have excellent anthelmintic activity. While a previous structure of the three-domain core region of Cry5B(112–698) had been reported, this structure lacked a key N-terminal extension critical to function. Here we report the structure of Cry5B(27–698) containing this N-terminal extension. This new structure adopts a distinct quaternary structure compared to the previous Cry5B(112–698) structure, and also exhibits a change in the conformation of residues 112–140 involved in linking the N-terminal extension to the three-domain core by forming a random coil and an extended α-helix. A role for the N-terminal extension is suggested based on a computational model of the tetramer with the conformation of residues 112–140 in its alternate α-helix conformation. Finally, based on the Cry5B(27–698) structure, site-directed mutagenesis studies were performed on Tyr495, which revealed that having an aromatic group or bulky group at this residue 495 is important for Cry5B toxicity.
Liposome was prepared using phosphatidylcholine (PC), and calcein, a fluorescent chemical, was simultaneously enclosed within the liposome (PC-Lipo). The stability of PC-Lipo in its ability to retain calcein was evaluated under various conditions. PC-Lipo lost stability at pH 6 and pH 11-13, but was stable in the range of pH 8.3-10. PC-Lipo was stable in the temperature range of 15-30 degrees C, but lost the stability acutely at 35 degrees C. Ionic strength, given as the concentration of NaCl, also affects its stability, and a higher concentration of NaCl, i.e., more than 150 mm, induced a higher leak of calcein from PC-Lipo. The optimal conditions to achieve stable PC-Lipo were employed to characterize the differences in pore formation with Bacillus thuringiensis Cry1Aa, Cry1Ab and Cry1Ac toxins. These toxins were reacted with PC-Lipo under these optimal conditions, and the affinity and maximum speed of Cry1Ab to form pores on PC-Lipo was shown to be highest. Here we show these conditions and evidence of the usefulness of PC-Lipo to investigate the mode of action of Cry1A toxins.
The pore-forming domain of Bacillus thuringiensis insecticidal Cry toxins is formed of seven amphipathic α-helices. Because pore formation is thought to involve conformational changes within this domain, the possible role of its interhelical loops in this crucial step was investigated with Cry9Ca double mutants, which all share the previously characterized R164A mutation, using a combination of homology modeling, bioassays and electrophysiological measurements. The mutations either introduced, neutralized or reversed an electrical charge carried by a single residue of one of the domain I loops. The ability of the 28 Cry9Ca double mutants to depolarize the apical membrane of freshly isolated Manduca sexta larval midguts was tested in the presence of either midgut juice or a cocktail of protease inhibitors because these conditions had been shown earlier to greatly enhance pore formation by Cry9Ca and its R164A single-site mutant. Most mutants retained toxicity toward neonate larvae and a pore-forming ability in the electrophysiological assay, which were comparable to those of their parental toxin. In contrast, mutants F130D, L186D and V189D were very poorly toxic and practically inactive in vitro. On the other hand, mutant E129A depolarized the midgut membrane efficiently despite a considerably reduced toxicity, and mutant Q192E displayed a reduced depolarizing ability while conserving a near wild-type toxicity. These results suggest that the conditions found in the insect midgut, including high ionic strength, contribute to minimizing the influence of surface charges on the ability of Cry9Ca and probably other B. thuringiensis toxins to form pores within their target membrane.
Cry toxins produced by Bacillus thuringiensis have been recognized as pore-forming toxins whose primary action is to lyse midgut epithelial cells in their target insect. In the case of the Cry1A toxins, a prepore oligomeric intermediate is formed after interaction with cadherin receptor. The Cry1A oligomer then interacts with glycosylphosphatidylinositol-anchored receptors. Two Manduca sexta glycosylphosphatidylinositol-anchored proteins, aminopeptidase (APN) and alkaline phosphatase (ALP), have been shown to bind Cry1Ab, although their role in toxicity remains to be determined. Detection of Cry1Ab binding proteins by ligand blot assay revealed that ALP is preferentially expressed earlier during insect development, because it was found in the first larval instars, whereas APN is induced later after the third larval instar. The binding of Cry1Ab oligomer to pure preparations of APN and ALP showed that this toxin structure interacts with both receptors with high affinity (apparent K(d) = 0.6 nM), whereas the monomer showed weaker binding (apparent K(d) = 101.6 and 267.3 nM for APN and ALP, respectively). Several Cry1Ab nontoxic mutants located in the exposed loop 2 of domain II or in beta-16 of domain III were affected in binding to APN and ALP, depending on their oligomeric state. In particular monomers of the nontoxic domain III, the L511A mutant did not bind ALP but retained APN binding, suggesting that initial interaction with ALP is critical for toxicity. Our data suggest that APN and ALP fulfill two roles. First APN and ALP are initial receptors promoting the localization of toxin monomers in the midgut microvilli before interaction with cadherin. Then APN and ALP function as secondary receptors mediating oligomer insertion into the membrane. However, the expression pattern of these receptors and the phenotype of L511A mutant suggest that ALP may have a predominant role in toxin action because Cry toxins are highly effective against the neonate larvae that is the target for pest control programs.
The toxicity and pore-forming ability of the Bacillus thuringiensis Cry9Ca insecticidal toxin, its single-site mutants, R164A and R164K, and the 55-kDa fragment resulting from its proteolytic cleavage at residue 164 were investigated using Manduca sexta neonate larvae and fifth-instar larval midgut brush border membrane vesicles, respectively. Neither the mutations nor the proteolytic cleavage altered Cry9Ca toxicity. Compared with Cry1Ac, Cry9Ca and its mutants formed large poorly selective pores in the vesicles. Pore formation was highly dependent on pH, however, especially for wild-type Cry9Ca and both mutants. Increasing pH from 6.5 to 10.5 resulted in an irregular step-wise decrease in membrane permeabilization that was not related to a change in the ionic selectivity of the pores. Pore formation was much slower with Cry9Ca and its derivatives, including the 55-kDa fragment, than with Cry1Ac and its rate was not influenced by the presence of protease inhibitors or a reducing agent.
Optical biosensor technology continues to be the method of choice for label-free, real-time interaction analysis. But when it comes to improving the quality of the biosensor literature, education should be fundamental. Of the 1413 articles published in 2008, less than 30% would pass the requirements for high-school chemistry. To teach by example, we spotlight 10 papers that illustrate how to implement the technology properly. Then we grade every paper published in 2008 on a scale from A to F and outline what features make a biosensor article fabulous, middling or abysmal. To help improve the quality of published data, we focus on a few experimental, analysis and presentation mistakes that are alarmingly common. With the literature as a guide, we want to ensure that no user is left behind.
Receptor binding studies were performed with I-125-labeled trypsin-activated insecticidal toxins, CryIA(a) and CryIA(c), from Bacillus thuringiensis on brush-border membrane vesicles (BBMV) prepared from Bombyx mori larval midgut. Bioassays were performed by gently force feeding B. mori with diluted toxins. CryIA(a) toxin (LD50; 0.002-mu-g) was 200 times more active against B. mori larvae than CryIA(c) toxin (LD50; 0.421-mu-g) and showed high-affinity saturable binding. The K(d) and the binding site concentration for CryIA(a) toxin were 3.5 nM and 7.95 pmol/mg, respectively. CryIA(c) toxin (K(d), 50.35 nM; B(max), 2.85 pmol/mg) did not demonstrate high-affinity binding to B. mori BBMV. Control experiments with CryIA(a) and CryIA(c) toxins revealed no binding to mouse small intestine BBMV and nonspecific binding to pig kidney BBMV. These data provide evidence that binding to a specific receptor on the membrane of midgut epithelial cells is an important determinant with respect to differences in insecticidal spectrum of insecticidal crystal proteins. To locate a B. mori receptor binding region on the CryIA(a) toxin, homologous and heterologous competition binding studies were performed with a set of mutant proteins which had previously been used to define the B. mori ''specificity domain'' on this toxin (Ge, A. Z., Shivarova, N. I., and Dean, D. H. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4037-4041). These mutant proteins have had regions of their genes reciprocally exchanged with the cryIA(c) gene. A B. mori receptor binding region on CryIA(a) toxin includes the amino-terminal portion of the hypervariable region, amino acids 332-450, which is identical to the previously described B. mori specificity determining region. These data provide direct evidence that delta-endotoxins contain a tract of amino acids that comprise a binding region and as a results determines the specificity of a toxin.
The structure of the delta-endotoxin from Bacillus thuringiensis subsp. tenebrionis that is specifically toxic to Coleoptera insects (beetle toxin) has been determined at 2.5 A resolution. It comprises three domains which are, from the N- to C-termini, a seven-helix bundle, a three-sheet domain, and a beta sandwich. The core of the molecule encompassing all the domain interfaces is built from conserved sequence segments of the active delta-endotoxins. Therefore the structure represents the general fold of this family of insecticidal proteins. The bundle of long, hydrophobic and amphipathic helices is equipped for pore formation in the insect membrane, and regions of the three-sheet domain are probably responsible for receptor binding.
Fluorophores processing a 6-acyl-2-dimethylaminonaphthalene moiety show fluorescence that is extremely sensitive to solvent polarity (Weber, G., and Farris, F. J. (1979) Biochemistry 18, 3075-3078). We have synthesized and characterized 6-acryloyl-2-dimethylaminonaphthalene (Acrylodan) which selectively labels thiol moieties in proteins. The quantum yield of this agent is markedly enhanced after reaction with thiols, and as expected, the fluorescent derivatives are very sensitive to dipolar perturbation from their environments. The usefulness of Acrylodan in the study of "hydrophobic" domains, conformational changes, and dipolar relaxation processes in proteins is demonstrated by measurements of fluorescence spectra and lifetimes of a mercaptoethanol adduct dissolved in different solvents and of adducts of this agent with parvalbumin, troponin C, papain, and carbonic anhydrase.
A peptide with a sequence corresponding to the highly conserved α-5 segment of the Cry δ-endotoxin family (amino acids 193-215 of Bacillus thuringiensis CryIIIA, was investigated with respect to its interaction with insect membranes, cytotoxicity in vitro towards Spodoptera frugiperda (Sf-9) cells, and its propensity to form ion channels in planar lipid membranes (PLMs). Selectively labelled analogues of α-5 at either the N-terminal amino acid or the ε-amine of its lysine, were used to monitor the interaction of the peptides with insect membranes. The fluorescent emission spectra of the 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD)-labelled α-5 peptides displayed a blue shift upon binding to insect (Spodoptera littoralis) mid-gut membranes, reflecting the relocation of the fluorescent probes to an environment of increased apolarity, i.e. within the lipidic constituent of the membrane. Moreover, midgut membrane-bound NBD-labelled α-5 peptides were protected from enzymic proteolysis. Functional characterization of α-5 has revealed that it is cytotoxic to Sf-9 insect cells, and that it forms ion channels in PLMs with conductances ranging from 30 to 1000 pS. A proline-substituted analogue of α-5 is less cytolytic and slightly more exposed to enzymic digestion. Molecular modelling utilizing simulated annealing via molecular dynamics suggests that a transbilayer pore may be formed by α-5 monomers that assemble to form a left-handed coiled coil of approximately parallel helices. These findings further support a role for α-5 in the toxic mechanism of δ-endotoxins, and assign α-5 as one of the transmembrane helices which form the toxic pore. The suggested role is consistent with the recent finding that cleavage of CryIVB δ-endotoxin in a loop between α-5 and α-6 is highly important for its larvicidal activity.
The pore-forming domain of Bacillus thuringiensis insecticidal CryIIIA δ-endotoxin contains two helices, α5 and α7, that are highly conserved within all different Cry 5-endotoxins. To gain information on the mode of action of δ-endotoxins, we have used a spectrofluorimetric approach and characterized the structure, the organization state, and the ability to self-assemble and to co-assemble within lipid membranes of α5 and α7. Circular dichroism (CD) spectroscopy revealed that α7 adopts a predominantly α-helical structure in methanol, similar to what has been found for α5, and consistent with its structure in the intact molecule. The hydrophobic moment of α7 is higher than that calculated for α5; however, α7 has a lesser ability to permeate phospholipids as compared to α5. Binding experiments with 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD)-labeled peptide demonstrated that α7 binds to phospholipid vesicles with a partition coefficient in the order of 104 M-1 similar to α5, but with reduced kinetics and in a noncooperative manner, as opposed to the fast kinetics and cooperativity found with α5. Resonance energy transfer measurements between fluorescently labeled pairs of donor (NBD)/acceptor (rhodamine) peptides revealed that, in their membrane-bound state, α5 self-associates but α7 does not, and that α5 coassembles with α7 but not with an unrelated membrane bound α-helical peptide. Furthermore, resonance energy transfer experiments, using α5 segments, specifically labeled in either the N-or C-terminal sides, suggest a parallel organization of α5 monomers within the membranes. Taken together the results are consistent with an umbrella model suggested for the pore forming activity of δ-endotoxin (Li, J., Caroll, J., and Ellar, D. J. (1991) Nature 353, 815-821), where α5 has transmembrane localization and may be part of the pore lining segment(s) while α7 may serve as a binding sensor that initiates the binding of the pore domain to the membrane.
CryIA(c) delta-endotoxin, a member of the CryI family of Bacillus thuringiensis insecticidal proteins, specifically recognizes and binds with high affinity to target proteins in the midgut of susceptible insects. Protein blots of Manduca sexta brush-border membranes probed with 125I-CryIA(c) identify a major binding protein of 120 kDa and a minor binding protein of 65 kDa. Monoclonal antibodies were raised against the 120-kDa toxin binding protein. Using isoelectric focusing and monoclonal antibodies (2B3, 8G1, and 12B8) 120- and 65-kDa brush-border proteins were isolated. Labeled CryIA(c) and monoclonal antibodies probed to blots of the affinity-selected proteins recognized the 120- and 65-kDa proteins. When reconstituted into phospholipid vesicles, antibody-selected proteins increased toxin binding (35%) and enhanced toxin-induced 86Rb+ release up to 1000-fold. The 120-kDa protein was identified as aminopeptidase N (EC 3.4.11.2). A CryIA(c)-sensitive phosphatase was also present in the 120/65-kDa protein mixture. These findings provide the first identification of B. thuringiensis toxin binding proteins, although confirmation is needed in vivo.
The Bacillus thuringiensis toxin-binding properties of midgut epithelial cells from two strains of Heliothis virescens were compared. One H. virescens strains (YHD2) which was selected against CryIAc toxin had over 10,000-fold resistance to CryIAc toxin relative to the susceptible strain and was cross-resistant to CryIAa and CryIAb. The second H. virescens strain (YDK) was susceptible to these toxins in the order CryIAc > CryIAb > CryIAa. Receptor-binding properties of CryIAa, CryIAb, and CryIAc toxins were compared between the susceptible and resistant strains. Saturation and competition-binding experiments were performed with brush border membrane vesicles prepared from midguts of the susceptible and resistant insects and 125I-labeled toxins. In the susceptible strain, saturable, specific, and high-affinity binding of all three toxins was observed. The relative binding-site concentration was directly correlated with toxicity (CryIAc > CryIAb > CryIAa). In the resistant strains, the binding affinities of CryIAb and CryIAc were similar to that observed with the susceptible strain and ony minor differences in binding-site concentration (Bmax) were observed. The major difference between the two strains was the total lack of binding of CryIAa toxin to the brush border membrane vesicles of the resistant strain. Heterologous competition-binding experiments and ligand blot analysis supported the hypothesis that there were multiple binding sites for the toxins. On the basis of results of the present study, we propose that alterations in binding proteins shared by all three toxins are a major factor in resistance. This suggests that not all receptors of CryIAc might be involved in toxic function.
Site-directed mutagenesis was used to examine the role of domain II, loop 2 residues,
368RRPFNIGI375, of Bacillus thuringiensis insecticidal protein CryIAb. Alanine substitution of residues
368RRP370, called B4, abolished potency toward Manduca sexta and Heliothis virescens, and the loss of toxicity was correlated directly to substantially reduced binding affinity to brush-border membrane vesicles
(BBMV) prepared from the target insect midguts. These results indicated that these positive charges might be essential to
orient the toxin to midgut receptor molecule(s). The role of residue Phe371 of CryIAb toxin to M. sexta was investigated by substituting a series of residues at this position. Irreversible binding and toxicity were affected significantly
by hydrophilic, aliphatic, and smaller side-chain residues such as Cys, Val, Leu, and Ser but not by Tyr or Trp. A hydrophobic
aromatic side-chain residue at position 371 was therefore essential for irreversible binding of CryIAb toxin in M. sexta. The role of residues 370PFNIGI375 of CryIAb toxin on H. virescens was also examined. Mutants D2 (deletion of residues 370-375), G374A (alanine substitution of Gly374), and I375A had reduced toxicity to H. virescens. In contrast to our findings with M. sexta, the reduction in toxicity of these mutants was correlated directly with loss of initial binding to H. virescens BBMV, indicating that these residues perform functionally distinct roles in binding and toxicity to different insects. In
ligand blots, CryIAb recognized a major 210-kDa peptide in M. sexta BBMV and a 170-kDa peptide in H. virescens BBMV.
To test whether the ability of Bacillus thuringiensis toxins to form pores in the midgut epithelial cell membrane of susceptible insects correlates with their in vivo toxicity, we measured the effects of different toxins on the electrical potential of the apical membrane of freshly isolated midguts from gypsy moth (Lymantria dispar) and silkworm (Bombyx mori) larvae. In the absence of toxin, the membrane potential, measured with a conventional glass microelectrode, was stable for up to 30 min. It was sensitive to the K+ concentration and the oxygenation of the external medium. Addition of toxins to which L. dispar is highly [CryIA(a) and CryIA(b)] or only slightly [CryIA(c) and CryIC] sensitive caused a rapid, irreversible, and dose-dependent depolarization of the membrane. CryIF, whose toxicity towards L. dispar is unknown, and CryIE, which is at best poorly active in vivo, were also active in vitro. In contrast, CryIB and CryIIIA, a coleopteran-specific toxin, had no significant effect. The basolateral-membrane potential was unaffected by CryIA(a) or CryIC when the toxin was applied to the basal side of the epithelium. In B. mori midguts, the apical-membrane potential was abolished by CryIA(a), to which silkworm larvae are susceptible, but CryIA(b) and CryIA(c); to which they are resistant, had no detectable effect. Although the technique discriminated between active and inactive toxins, the concentration required to produce a given effect varied much less extensively than the sensitivity of gypsy moth larvae, suggesting that additional factors influence the toxins' level of toxicity in vivo.
The role of the third domain of CryIAa, a Bacillus thuringiensis insecticidal toxin, in toxin-induced membrane permeabilization in a receptor-free environment was investigated. Planar lipid bilayer experiments were conducted with the parental toxin and five proteins obtained by site-directed mutagenesis in block 4, an arginine-rich, highly conserved region of the protein. Four mutants were constructed by replacing the first arginine in position 21 by a lysine (R521K), a glutamine (R521Q), a histidine (R521H), or a glutamic acid (R521E). A fifth mutant was obtained by replacing the fourth arginine by a lysine (R527K). Like CryIAa, the mutants formed cation-selective channels. A limited but significant reduction in channel conductance was observed for all mutants except R521H. The effect was more dramatic for the voltage dependence of the channels formed by R521K and R521Q, which was reversed compared to that of the parental toxin. This study provides the first direct evidence of a functional role for domain III in membrane permeabilization. Our results suggest that residues of the positive arginine face of block 4 interact with domain I, the putative pore-forming region of CryIAa.
During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism's pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit.
The mode of action of Bacillus thuringiensis insecticidal proteins is not well understood. Based on analogies with other bacterial toxins and ion channels, we hypothesized that charged amino acids in helix 4 of the Cry1Aa toxin are critical for toxicity and ion channel function. Using Plutella xylostella as a model target, we analyzed responses to Cry1Aa and eight proteins with altered helix 4 residues. Toxicity was abolished in five charged residue mutants (E129K, R131Q, R131D, D136N, D136C), however, two charged (R127E and R127N) and one polar (N138C) residue mutant retained wild-type toxicity. Compared with Cry1Aa and toxic mutants, nontoxic mutants did not show greatly reduced binding to brush border membrane vesicles, but their ion channel conductance was greatly reduced in planar lipid bilayers. Substituted cysteine accessibility tests showed that in situ restoration of the negative charge of D136C restored conductance to wild-type levels. The results imply that charged amino acids on the Asp-136 side of helix 4 are essential for toxicity and passage of ions through the channel. These results also support a refined version of the umbrella model of membrane integration in which the side of helix 4 containing Asp-136 faces the aqueous lumen of the ion channel.
Bacillus thuringiensis delta-endotoxins insert into the brush border membranes of insect larval cells to form ion channels. A possible interaction of these toxins with a cytoplasmic component was examined by preloading vesicles from insect larval cells with protease K followed by incubation with toxin. There was no evidence for toxin antigens smaller than the intact toxin in extracts of solubilized vesicles, nor was there an effect of the inclusion of protease K on either of two functional properties, the formation of toxin aggregates or of ion pores. These toxins, physically and functionally, appear to be confined to the membrane.
After activation, Bacillus thuringiensis (Bt) insecticidal toxin forms pores in larval midgut epithelial cell membranes, leading to host death. Although the crystal structure of the soluble form of Cry1Aa has been determined, the conformation of the pores and the mechanism of toxin interaction with and insertion into membranes are still not clear. Here we show that Cry1Aa spontaneously inserts into lipid mono- and bilayer membranes of appropriate compositions. Fourier Transform InfraRed spectroscopy (FTIR) indicates that insertion is accompanied by conformational changes characterized mainly by an unfolding of the beta-sheet domains. Moreover, Atomic Force Microscopy (AFM) imaging strongly suggests that the pores are composed of four subunits surrounding a 1.5 nm diameter central depression.
Lipid rafts are characterized by their insolubility in nonionic detergents such as Triton X-100 at 4 °C. They have been studied
in mammals, where they play critical roles in protein sorting and signal transduction. To understand the potential role of
lipid rafts in lepidopteran insects, we isolated and analyzed the protein and lipid components of these lipid raft microdomains
from the midgut epithelial membrane of Heliothis virescens andManduca sexta. Like their mammalian counterparts, H. virescens and M. sexta lipid rafts are enriched in cholesterol, sphingolipids, and glycosylphosphatidylinositol-anchored proteins. In H. virescens and M. sexta, pretreatment of membranes with the cholesterol-depleting reagent saponin and methyl-β-cyclodextrin differentially disrupted
the formation of lipid rafts, indicating an important role for cholesterol in lepidopteran lipid rafts structure. We showed
that several putativeBacillus thuringiensis Cry1A receptors, including the 120- and 170-kDa aminopeptidases from H. virescens and the 120-kDa aminopeptidase from M. sexta, were preferentially partitioned into lipid rafts. Additionally, the leucine aminopeptidase activity was enriched approximately
2–3-fold in these rafts compared with brush border membrane vesicles. We also demonstrated that Cry1A toxins were associated
with lipid rafts, and that lipid raft integrity was essential for in vitro Cry1Ab pore forming activity. Our study strongly suggests that these microdomains might be involved in Cry1A toxin aggregation
and pore formation.
The cadherin protein Bt-R1a is a receptor for Bacillus thuringiensis Cry1A toxins in Manduca sexta. Cry1Ab toxin is reported to bind specific epitopes located in extracellular cadherin repeat (CR) 7 and CR11 on Bt-R1 (Gomez, B., Miranda-Rios, J., Riudino-Pinera, E., Oltean, D. I., Gill, S. S., Bravo, A., and Soberon, M. (2002) J. Biol. Chem. 277, 30137–30143; Dorsch, J. A., Candas, M., Griko, N., Maaty, W., Midboe, E., Vadlamudi, R., and Bulla, L. (2002) Insect Biochem. Mol. Biol. 32, 1025–1036). We transiently expressed CR domains of Bt-R1a in Drosophila melanogaster Schneider 2 (S2) cells as fusion peptides between a signal peptide and a terminal region that included membrane-proximal,
membrane-spanning, and cytoplasmic domains. A domain consisting of CR11 and 12 was the minimal 125I-Cry1Ab binding region detected under denaturing conditions. Only CR12 was essential for Cry1Ab binding and cytotoxicity
to S2 cells when tested under native conditions. Under these conditions expressed CR12 bound 125I-Cry1Ab with high affinity (Kcom = 2.9 nm). Flow cytometry assays showed that expression of CR12 conferred susceptibility to Cry1Ab in S2 cells. Derivatives of Bt-R1a with separate deletions of CR7, 11, and 12 were expressed in S2 cells. Only deletion of CR12 caused loss of Cry1Ab binding
and cytotoxicity. These results demonstrate that CR12 is the essential Cry1Ab binding component on Bt-R1 that mediates Cry1Ab-induced cytotoxicity.
The specific role of cadherin receptors in cytotoxicity involving Cry toxins of Bacillus thuringiensis and their interactions with cell membrane has not been defined. To elucidate the involvement of toxin-membrane and toxin-receptor interactions in cytotoxicity, we established a cell-based system utilizing High Five insect cells stably expressing BT-R1, the cadherin receptor for Cry1Ab toxin. Cry1Ab toxin is incorporated into cell membrane in both oligomeric and monomeric form. Monomeric toxin binds specifically to BT-R1 whereas incorporation of oligomeric toxin is nonspecific and lipid dependent. Toxin oligomers in the cell membrane do not produce lytic pores and do not kill insect cells. Rather, cell death correlates with binding of the Cry1Ab toxin monomer to BT-R1, which apparently activates a Mg2+-dependent cellular signaling pathway.
The Bacillus thuringiensis toxin-binding properties of midgut epithelial cells from two strains of Heliothis virescens were compared. One H. virescens strains (YHD2) which was selected against CryIAc toxin had over 10,000-fold resistance to CryIAc toxin relative to the susceptible strain and was cross-resistant to CryIAa and CryIAb. The second H. virescens strain (YDK) was susceptible to these toxins in the order CryIAc > CryIAb > CryIAa. Receptor-binding properties of CryIAa, CryIAb, and CryIAc toxins were compared between the susceptible and resistant strains. Saturation and competition-binding experiments were performed with brush border membrane vesicles prepared from midguts of the susceptible and resistant insects and 125I-labeled toxins. In the susceptible strain, saturable, specific, and high-affinity binding of all three toxins was observed. The relative binding-site concentration was directly correlated with toxicity (CryIAc > CryIAb > CryIAa). In the resistant strains, the binding affinities of CryIAb and CryIAc were similar to that observed with the susceptible strain and ony minor differences in binding-site concentration (Bmax) were observed. The major difference between the two strains was the total lack of binding of CryIAa toxin to the brush border membrane vesicles of the resistant strain. Heterologous competition-binding experiments and ligand blot analysis supported the hypothesis that there were multiple binding sites for the toxins. On the basis of results of the present study, we propose that alterations in binding proteins shared by all three toxins are a major factor in resistance. This suggests that not all receptors of CryIAc might be involved in toxic function.
Deletion of amino acid residues 370 to 375 (D2) and single alanine substitutions between residues 371 and 375 (FNIGI) of lepidopteran-active Bacillus thuringiensis CryIAb delta-endotoxin were constructed by site-directed mutagenesis techniques. All mutants, except that with the I-to-A change at position 373 (I373A), produced delta-endotoxin as CryIAb and were stable upon activation either by Manduca sexta gut enzymes or by trypsin. Mutants D2, F371A, and G374A lost most of the toxicity (400 times less) for M. sexta larvae, whereas N372A and I375A were only 2 times less toxic than CryIAb. The results of homologous and heterologous competition binding assays to M. sexta midgut brush border membrane vesicles (BBMV) revealed that the binding curves for all mutant toxins were similar to those for the wild-type toxin. However, a significant difference in irreversible binding was observed between the toxic (CryIAb, N372A, and I375A) and less-toxic (D2, F371A, and G374A) proteins. Only 20 to 25% of bound, radiolabeled CryIAb, N372A, and I375A toxins was dissociated from BBMV, whereas about 50 to 55% of the less-toxic mutants, D2, F371A, and G374A, was dissociated from their binding sites by the addition of excess nonlabeled ligand. Voltage clamping experiments provided further evidence that the insecticidal property (inhibition of short-circuit current across the M. sexta midgut) was directly correlated to irreversible interaction of the toxin with the BBMV. We have also shown that CryIAb and mutant toxins recognize 210- and 120-kDa peptides in ligand blotting. Our results imply that mutations in residues 370 to 375 of domain II of CrylAb do not affect overall binding but do affect the irreversible association of the toxin to the midgut columnar epithelial cells of M. sexta.
This chapter provides an overview of the biology of Bacillus thuringiensis (Bt). The chapter examines how the recently solved X-ray crystal structure of one Bt toxin allows to model the mode of action of a whole family of related protein toxins, and how a knowledge of the physiology of the insect targets of Bt toxins is needed for a better understanding of the toxic mechanism. Bacillus thuringiensis (Bt), a family of bacteria which make insecticidal proteins, accounts for 90–95% of the insect biocontrol market. This chapter illustrates that Bt is the name given to a family of bacteria found throughout the world. Paradoxically, CryIIIA, the only Bt toxin whose three-dimensional structure has been solved, kills very small insects, and its insolubility at neutral pH makes it difficult to assay in vitro. The crystal structure of CryIIIA permits site-directed mutagenesis and segment swapping experiments to be designed in a rational manner in order to identify the functional domains of the toxins. This chapter explores one attractive prospect that can be the replacement of the specificity domain with a binding domain directed to a target of choice, opening up the possibility of “designer” pesticides or a new class of immunotoxins. Characterization of toxin receptors and investigation of the mechanism of toxin synergism may assist in strategies for resistance management, a vital concern if Bt is to maintain its important position as the most extensively used biological insecticide.
The midgut of the tobacco hornworm (Manduca sexta) undergoes structural reorganization during the last larval stadium. Changes in midgut active ion transport and metabolism during this transition were studied by monitoring short-circuit current, maximal enzyme activities, and mitochondrial respiration. The short-circuit current of midguts from feeding caterpillars was over seven times higher than that of wandering larvae. No short-circuit current was detected in midguts from prepupae, whereas the midguts of pupae had a small but significant level of active ion transport. Paralleling the decline in active ion transport during larval development was the decline in the activity of citrate synthase, a key enzyme in oxidative metabolism. Aerobic metabolism was explored further by monitoring the oxidation of substrates by mitochondria isolated from the midguts of feeding and wandering larvae. Oxidation of all substrates, except ascorbate, was lower in mitochondria isolated from wandering larvae. These results indicate that the decline in active ion transport during larval development may be caused by a decline in mitochondrial energy production. Furthermore, this study demonstrates that the highly aerobic larval midgut is reorganized into a pupal epithelium with low rates of ion transport and oxidative metabolism.
Interaction of delta -endotoxin and its proteolytic fragments with phospholipid vesicles was studied using electron microscopy, scanning microcalorimetry, and limited proteolysis. It was shown that native protein destroys liposomes. The removal of 4 N-terminal alpha -helices and the extreme 56 C-terminal amino acid residues did not affect this ability. The results obtained by limited proteolysis of delta -endotoxin bound to lipid vesicles show essential conformational changes in three or four N-terminal helices and in the C-terminal region. The calorimetric method used in this study provides a unique possibility for the validation of existing models of protein binding and for a more accurate determination of the regions where conformational changes take place. It was found that the binding of the protein to model liposomes does not alter its structure in the regions starting with the fourth alpha -helix of domain I. This can be concluded from the fact that the activation energy of denaturation of the protein remains unchanged upon its binding to the phospholipid membranes. A new structural model has been proposed which agrees with the data obtained.
Bacillus thuringiensis δ-endotoxins (Cry proteins), are widely used for insect control and plant protection. They are water-soluble proteins that insert into membranes forming ion channels. In most Cry toxins α-helix 2 is broken by a highly conserved proline residue (Pro70 in Cry1Ab), generating a broken-helix motif. The flexibility of the motif was altered through site-directed mutagenesis. It was found that increasing the flexibility of the motif decreased the stability, the ion transport ability and the toxicity of the protein. By removing the broken-helix motif, the biological properties were restored to a wild type level.
The pore-forming domain of Bacillus thuringiensis insecticidal CryIIIA -endotoxin contains two helices, α5 and α7, that are highly conserved within all different Cry -endotoxins. To gain information on the mode of action of -endotoxins, we have used a spectrofluorimetric approach and characterized the structure, the organization state, and the
ability to self-assemble and to co-assemble within lipid membranes of α5 and α7. Circular dichroism (CD) spectroscopy revealed
that α7 adopts a predominantly α-helical structure in methanol, similar to what has been found for α5, and consistent with
its structure in the intact molecule. The hydrophobic moment of α7 is higher than that calculated for α5; however, α7 has
a lesser ability to permeate phospholipids as compared to α5. Binding experiments with 7-nitrobenz-2-oxa-1,3-diazole-4-yl
(NBD)-labeled peptide demonstrated that α7 binds to phospholipid vesicles with a partition coefficient in the order of 104M similar to α5, but with reduced kinetics and in a noncooperative manner, as opposed to the fast kinetics and cooperativity
found with α5. Resonance energy transfer measurements between fluorescently labeled pairs of donor (NBD)/acceptor (rhodamine)
peptides revealed that, in their membrane-bound state, α5 self-associates but α7 does not, and that α5 coassembles with α7
but not with an unrelated membrane bound α-helical peptide. Furthermore, resonance energy transfer experiments, using α5 segments,
specifically labeled in either the N- or C-terminal sides, suggest a parallel organization of α5 monomers within the membranes.
Taken together the results are consistent with an umbrella model suggested for the pore forming activity of -endotoxin (Li, J., Caroll, J., and Ellar, D. J.(1991) Nature 353, 815-821), where α5 has transmembrane localization and may be part of the pore lining segment(s) while α7 may serve as a
binding sensor that initiates the binding of the pore domain to the membrane.
The crystal δ-endotoxins of Bacillus thuringiensis (Bt) are a family of insecticidal proteins which have been known for some time to kill insects by lysing their gut epithelial cells, but the precise molecular mechanism of toxicity has remained elusive. The recent publication of the crystal structure of a Bt δ-endotoxin has made it possible for us to model the molecular events that occur as the toxin binds to its receptor and inserts into the membrane to form a pore. Using our knowledge of insect gut physiology, we can also predict the effect on the insect of the formation of a toxic pore. We present a new model to explain the events that occur in the insect gut during toxin action.
The activated 65 kDa lepidopteran-specific CryIA(a) toxin from the commercially most important strainBacillus thuringiensisvar.kurstakiHD-1 has been investigated by X-ray diffraction and for its ability to form channels in planar lipid bilayers. Its three-dimensional structure has been determined by a multiple isomorphous replacement method and refined at 2.25 Å resolution to anR-factor of 0.168 for data withI2δ(I). The toxin is made of three distinct domains. The N-terminal domain is a bundle of eight α-helices with the central, relatively hydrophobic helix surrounded by amphipathic helices. The middle and C-terminal domains contain mostly β-sheets. Comparison with the structure of CryIIIA, a coleopteran-specific toxin, shows that although the fold of these two proteins is similar, there are significant structural differences within domain II. This finding supports the conclusions from genetic studies that domain II is involved in recognition and binding to cell surface receptors. The distribution of electrostatic potential on the surface of the molecule is non-uniform and identifies one side of the α-helical domain as negatively charged. The predominance of arginine residues as basic residues ensures that the observed positive charge distribution is also maintained in the highly alkaline environment found in the lepidopteran midgut. Structurally important salt bridges that are conserved across Cry sequences were identified and their possible role in toxin action was postulated. In planar lipid bilayers, CryIA(a) forms cation-selective channels, whose conductance is significantly smaller than that reported for CryIIIA but similar to those of other Cry toxins.
The purification procedure of the toxic fraction from the δ-endotoxin produced by Bacillus thuringiensis var. entomocidus was considerably shortened and simplified by combining the solubilization in a high pH (10.0) solution with glycine, opening SS bonds with dithiothreitol, and releasing hydrophobic connections with the detergents Triton N-101 and sodium cholate. The subsequent fractionation was immediately continued (without dialysis or concentration) on a Sepharose 6B column, yielding the active fraction designated C. Fraction C was then passed through an octyl Sepharose 4B column, yielding the active fraction designated I, followed by gel filtration on a Sepharose 6B column yielding the active fraction designated CI. All treatments were done with the same buffer solution at 4°C. Column elution was with the same buffer but with 0.075% detergents. The purity of the fraction CI was apparent by its appearance as a well-defined band on polyacrylamide gel isoelectric focusing at pH 6.1, accompanied by a small faint band.A sensitive method for evaluating the toxicity of endotoxin fractions on an isolated midgut system (from larvae of Spodoptera littoralis) was developed. The system is based on measuring the activity of the enzyme, reduced glutathione S-transferase, released to the medium from epithelial cells ruptured by the toxin. Fraction CI, Mr 64,000, was toxic to the epithelial cells of the isolated midgut in the absence of the peritrophic membrane. When the detergents were removed by dialysis, the active protein formed high molecular weight aggregates due to hydrophobic interactions. The passage of those aggregates to the sensitive epithelial cells of the midgut was prevented by the peritrophic membrane.
We examined the abilities of two Bacillus thuringiensis insecticidal toxins, CryIAa and CryIAc, prepared from Escherichia coli-cloned gene products, to inhibit short-circuit current in midgut epithelia of Bombyx mori. Voltage-clamp studies were conducted on isolated midguts, measuring the inhibition of short-circuit current (ISC) by trypsin-activated toxins. Three bathing solutions were compared and the medium of Chamberlin [(1990) J. exp. Biol. 150, 467–471] was found to maintain the highest ISC for the longest time. For CryIAa, a concentration range between 0.33 and 8.0 ng/ml resulted in inhibition of ISC at the rates of −0.91 μA/min (lag time, 9 min) and −7.13 μA/min (lag time, 4 min) respectively, showing a correlation between toxin concentration and inhibitory response. Concentrations greater than 1.6 ng/ml showed diminishing additional effects on the ISC response, indicating an approach to saturation. The lag times decreased with increasing concentration of toxin applied. For CryIAc, the lowest concentration that gave a response was 3.2 ng/ml (slope, −0.31 μA/min; lag time, 2 min). There also was a linear correlation between toxin concentration and response for CryIAc, but effective concentrations of CryIAc were approximately 2 orders of magnitude greater than those of CryIAa.
1.1. Brush border membrane vesicles (BBMV) were prepared from midguts of Pieris brassicae larvae by Mg/EGTA precipitation and differential centrifugation.2.2. Their morphology, polypeptide composition, and marker enzyme enrichment was similar to BBMV from other larval lepidopteran midguts.3.3. In the presence of an inwardly directed KSCN gradient, these BBMV transiently accumulated alanine, phenylalanine, histidine, lysine, or gultamic acid.4.4. Initial uptake rates for neutral and basic amino acids were similar in the presence of either a potassium or a sodium gradient.5.5. Initial uptake of glutamic acid was much more efficient in the presence of a sodium gradient.
Receptor binding studies were performed with 125I-labeled trypsin-activated insecticidal toxins, CryIA(a) and CryIA(c), from Bacillus thuringiensis on brush-border membrane vesicles (BBMV) prepared from Bombyx mori larval midgut. Bioassays were performed by gently force feeding B. mori with diluted toxins. CryIA(a) toxin (LD50; 0.002 micrograms) was 200 times more active against B. mori larvae than CryIA(c) toxin (LD50; 0.421 micrograms) and showed high-affinity saturable binding. The Kd and the binding site concentration for CryIA(a) toxin were 3.5 nM and 7.95 pmol/mg, respectively. CryIA(c) toxin (Kd, 50.35 nM; Bmax, 2.85 pmol/mg) did not demonstrate high-affinity binding to B. mori BBMV. Control experiments with CryIA(a) and CryIA(c) toxins revealed no binding to mouse small intestine BBMV and nonspecific binding to pig kidney BBMV. These data provide evidence that binding to a specific receptor on the membrane of midgut epithelial cells is an important determinant with respect to differences in insecticidal spectrum of insecticidal crystal proteins. To locate a B. mori receptor binding region on the CryIA(a) toxin, homologous and heterologous competition binding studies were performed with a set of mutant proteins which had previously been used to define the B. mori "specificity domain" on this toxin (Ge, A. Z., Shivarova, N. I., and Dean, D. H. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4037-4041). These mutant proteins have had regions of their genes reciprocally exchanged with the cryIA(c) gene. A B. mori receptor binding region on CryIA(a) toxin includes the amino-terminal portion of the hypervariable region, amino acids 332-450, which is identical to the previously described B. mori specificity determining region. These data provide direct evidence that delta-endotoxins contain a tract of amino acids that comprise a binding region and as a results determines the specificity of a toxin.
The protein delta-endotoxins of Bacillus thuringiensis are a commercially and environmentally important class of highly specific insecticides. From an alignment of their sequences, certain structural and functional domains can be inferred which may shed light on the mode of action of these toxins.
Deletion of amino acid residues 370 to 375 (D2) and single alanine substitutions between residues 371 and 375 (FNIGI) of lepidopteran-active Bacillus thuringiensis CryIAb delta-endotoxin were constructed by site-directed mutagenesis techniques. All mutants, except that with the I-to-A change at position 373 (I373A), produced delta-endotoxin as CryIAb and were stable upon activation either by Manduca sexta gut enzymes or by trypsin. Mutants D2, F371A, and G374A lost most of the toxicity (400 times less) for M. sexta larvae, whereas N372A and I375A were only 2 times less toxic than CryIAb. The results of homologous and heterologous competition binding assays to M. sexta midgut brush border membrane vesicles (BBMV) revealed that the binding curves for all mutant toxins were similar to those for the wild-type toxin. However, a significant difference in irreversible binding was observed between the toxic (CryIAb, N372A, and I375A) and less-toxic (D2, F371A, and G374A) proteins. Only 20 to 25% of bound, radiolabeled CryIAb, N372A, and I375A toxins was dissociated from BBMV, whereas about 50 to 55% of the less-toxic mutants, D2, F371A, and G374A, was dissociated from their binding sites by the addition of excess nonlabeled ligand. Voltage clamping experiments provided further evidence that the insecticidal property (inhibition of short-circuit current across the M. sexta midgut) was directly correlated to irreversible interaction of the toxin with the BBMV. We have also shown that CryIAb and mutant toxins recognize 210- and 120-kDa peptides in ligand blotting. Our results imply that mutations in residues 370 to 375 of domain II of CrylAb do not affect overall binding but do affect the irreversible association of the toxin to the midgut columnar epithelial cells of M. sexta.
A 120 kDa glycoprotein in the larval midgut membrane of the lepidopteran Manduca sexta, previously identified as a putative receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin, has been purified by a combination of protoxin affinity chromatography and anion exchange chromatography. In immunoblotting experiments, the purified glycoprotein has the characteristics predicted of the receptor: it binds CrylA(c) toxin in the presence of GlcNAc but not GalNAc; it binds the lectin SBA; but it does not bind CrylB toxin. N-terminal and internal amino acid sequences obtained from the protein show a high degree of similarity with the enzyme aminopeptidase N (EC 3.4.11.2). When assayed for aminopeptidase activity, purified receptor preparations were enriched 5.3-fold compared to M. sexta brush border membrane vesicles. We propose that the receptor for CrylA(c) toxin in the brush border membrane of the lepidopteran M. sexta is the metalloprotease aminopeptidase N.
Bacillus thuringiensis delta-endotoxins (Cry toxins) are insecticidal proteins of approximately 65 kDa in the proteolytically processed and active form. The structure of one of these toxins, CryIIIA, has been determined by Li et al. [Li, J., Carroll, J. & Ellar, D. J. (1991) Nature (London) 353, 815-821] and contains three domains. It is believed that other delta-endotoxins adopt similar three-dimensional structure. Li et al. proposed that the first domain is the membrane pore-forming domain. Previous work from our laboratory has shown that the second domain is the receptor binding domain, but the function of the third domain is unclear. Site-directed mutagenesis was used to convert the "arginine face" of one of five highly conserved regions, QRYRVRIRYAS of CryIAa (residues 525-535), to selected other residues. This sequence corresponds to the beta-sheet 17 of CryIIIA in the third domain. Mutations in the second and third arginine positions resulted in structural alterations in the protein and were poorly expressed in Escherichia coli. Toxins from genes mutated to replace lysine for the first and fourth arginines were unaltered in expression and structure, as measured by trypsin activation, CD spectra, and receptor binding, but were substantially reduced in their insecticidal properties and inhibition of short circuit current across Bombyx mori midguts. It is proposed that this region plays a role in toxin function as an ion channel.
A series of mutant Bacillus thuringiensis CryIAa delta-endotoxin proteins was prepared by replacing the first, second, and last arginine residues of the conserved third-domain sequence, R-521 YRVRIR-527, with other amino acids. The stable mutant proteins were bioassayed against Bombyx mori larvae and found to all be approximately half as active as wild-type CryIAa. The toxins were also tested by means of a light-scattering assay for their ability to increase permeability of larval B. mori midgut brush border membrane vesicles. Three of the mutant toxins were as active as the wild-type toxin in the vesicle permeability assay.
Integral membrane proteins have recently been shown to recognize and interact with other proteins within the membrane, either mimicking or altering their function, and with the lipid bilayer itself, resulting in a reorganization of native membrane protein. Membrane proteins are difficult to study using conventional methods such as X-ray crystallography, and so both synthesized and naturally occurring segments of membrane proteins have been used in the assessment of the mechanisms involved in their structure and organization.
Disulfide bridges were introduced into CrylAa, a Bacillus thuringiensis lepidopteran toxin, to stabilize different protein domains including domain I alpha-helical regions thought to be involved in membrane integration and permeation. Bridged mutants could not form functional ion channels in lipid bilayers in the oxidized state, but upon reduction with beta-mercaptoethanol, regained parental toxin channel activity. Our results show that unfolding of the protein around a hinge region linking domain I and II is a necessary step for pore formation. They also suggest that membrane insertion of the hydrophobic hairpin made of alpha-helices 4 and 5 in domain I plays a critical role in the formation of a functional pore.
A purified, GPI-linked receptor complex isolated from Manduca sexta midgut epithelial cells was reconstituted in planar lipid bilayers. CryIAa, CryIAc and CryIC, three Bacillus thuringiensis insecticidal proteins, formed channels at much lower doses (0.33-1.7 nM) than in receptor-free membranes. The non-toxic protein CryIB also formed channels, but at doses exceeding 80 nM. The channels of CrylAc, the most potent toxin against M. sexta, rectified the passage of cations. All other toxin channels displayed linear current-voltage relationships. Therefore, reconstituted Cry receptors catalyzed channel formation in phospholipid membranes and, in two cases, were involved in altering their biophysical properties.
BT-R1, the Manduca sexta midgut receptor for the crystal toxin Cry1Ab produced by Bacillus thuringiensis ssp. berliner, was partly purified by gel filtration from M. sexta brush border membrane vesicles in the presence of the detergent CHAPS. Fractions containing BT-R1 were tested for their stability against degradation as indicated by retention of Cry1Ab binding on ligand blots. At 4 degrees C and pH 7.4 in the presence of Ca2+, BT-R1 was stable for up to 48 h but a 65% loss of binding was observed after 100 h. Under the same conditions, no loss of binding was observed in the presence of EGTA after 100 h. Cry1Ab binding decreased markedly as pH increased from 6 to 10 for incubations of 24 h at 4 degrees C. Increasing the temperature of incubation from 4 to 37 degrees C also decreased Cry1Ab binding. Neither metal ions nor free sulfhydryl groups are involved in Cry1Ab binding to BT-R1. A trypsin-like, metal-ion-dependent proteolytic activity co-eluted with BT-R1 during gel filtration. This endoproteolytic activity was unaltered by the addition of Cry1Ab. BT-R1 did not co-elute with peaks of aminopeptidase, alkaline phosphatase, alpha-glucosidase, beta-glucosidase and beta-galactosidase activities. When BT-R1 in the gel filtration fraction was further purified on a Mono Q anion exchange column, partial separation of the trypsin-like activity from BT-R1 was observed. BT-R1 could be removed from the appropriate Mono Q fraction by immunoprecipitation with only a slight decrease in this activity. These results demonstrate that there is no copurification of BT-R1 and these enzymes and that BT-R1 is unlikely to form complexes with them. Binding of Cry1Aa and Cry1Ac to BT-R1 in gel filtration fractions is similar to that of Cry1Ab, indicating that BT-R1 may be the high-affinity receptor for the Cry1A toxins. Binding of Cry1Ab to a 120 kDa protein has not been observed in this study.
The aim of this study was to elucidate the mechanism of membrane insertion and the structural organization of pores formed by Bacillus thuringiensis delta-endotoxin. We determined the relative affinities for membranes of peptides corresponding to the seven helices that compose the toxin pore-forming domain, their modes of membrane interaction, their structures within membranes, and their orientations relative to the membrane normal. In addition, we used resonance energy transfer measurements of all possible combinatorial pairs of membrane-bound helices to map the network of interactions between helices in their membrane-bound state. The interaction of the helices with the bilayer membrane was also probed by a Monte Carlo simulation protocol to determine lowest-energy orientations. Our results are consistent with a situation in which helices alpha4 and alpha5 insert into the membrane as a helical hairpin in an antiparallel manner, while the other helices lie on the membrane surface like the ribs of an umbrella (the "umbrella model"). Our results also support the suggestion that alpha7 may serve as a binding sensor to initiate the structural rearrangement of the pore-forming domain.
During sporulation, Bacillus thuringiensis produces crystalline inclusions comprised of a mixture of δ-endotoxins. Following ingestion by insect larvae, these inclusion proteins are solubilized, and the protoxins are converted to toxins. These bind specifically to receptors on the surfaces of midgut apical cells and are then incorporated into the membrane to form ion channels. The steps required for toxin insertion into the membrane and possible oligomerization to form a channel have been examined. When bound to vesicles from the midguts of Manduca sexta larvae, the Cry1Ac toxin was largely resistant to digestion with protease K. Only about 60 amino acids were removed from the Cry1Ac amino terminus, which included primarily helix α1. Following incubation of the Cry1Ab or Cry1Ac toxins with vesicles, the preparations were solubilized by relatively mild conditions, and the toxin antigens were analyzed by immunoblotting. In both cases, most of the toxin formed a large, antigenic aggregate of ca. 200 kDa. These toxin aggregates did not include the toxin receptor aminopeptidase N, but interactions with other vesicle components were not excluded. No oligomerization occurred when inactive toxins with mutations in amphipathic helices (α5) and known to insert into the membrane were tested. Active toxins with other mutations in this helix did form oligomers. There was one exception; a very active helix α5 mutant toxin bound very well to membranes, but no oligomers were detected. Toxins with mutations in the loop connecting helices α2 and α3, which affected the irreversible binding to vesicles, also did not oligomerize. There was a greater extent of oligomerization of the Cry1Ac toxin with vesicles from the Heliothis virescens midgut than with those from the M. sexta midgut, which correlated with observed differences in toxicity. Tight binding of virtually the entire toxin molecule to the membrane and the subsequent oligomerization are both important steps in toxicity.
Staphylococcal alpha-toxin forms heptameric pores that render membranes permeable for monovalent cations. The pore is formed by an amphipathic beta-barrel encompassing amino acid residues 118-140 of each subunit of the oligomer. Human fibroblasts are susceptible to alpha-toxin but are able to repair the membrane lesions. Thereby, toxin oligomers remain embedded in the plasma membrane and exposed to the extracellular medium. In this study, we sought to detect structural changes occurring in the pore-forming sequence during lesion repair. Single cysteine substitution mutants were labelled with the environmentally sensitive fluorochrome acrylodan and, after mixing with wild-type toxin, incorporated into hybrid heptamers on fibroblast membranes. Formation of the lipid-inserted beta-barrel was accompanied by characteristic fluorescence emission shifts. After lesion repair, the environment of the residues at the outer surface of the beta-barrel remained unchanged, indicating continued contact with lipids. However, the labelled residues oriented towards the channel lumen underwent a green to blue shift in fluorescence, indicating reduced exposure to water. Pore closure proceeded in the presence of calmodulin inhibitors and of microtubule disruptors; however, it was prevented by cytochalasin D and by inhibitors of lipid metabolism. Our findings reveal the existence of a novel mechanism of membrane repair that may consist in constriction of the inserted proteinaceous pore within the lipid bilayer.
We used site-directed mutagenesis to modify the Bacillus thuringiensis cry3A gene in amino acid residues 350-354. Two mutant toxins, A1 (R(345)A,Y(350)F,Y(351)F) and A2 (R(345)A,DeltaY(350), DeltaY(351)), showed significantly improved toxicity against Tenebrio molitor (yellow mealworm). The mutant toxin A1 was also more potent against both Leptinotarsa decemlineata (Colorado potato beetle) and Chrysomela scripta (cottonwood leaf beetle), while A2 displayed enhanced toxicity only in L. decemlineata. Competitive binding assays of L. decemlineata brush border membrane vesicles (BBMV) revealed that binding affinities for the A1 and A2 mutant toxins were ca. 2.5-fold higher than for the wild-type Cry3 toxin. Similar binding assays with C. scripta BBMV revealed a ca. 5-fold lower dissociation rate for the A1 mutant as compared to that of Cry3A.
Bacillus thuringiensis delta-endotoxins (Cry proteins), are widely used for insect control and plant protection. They are water-soluble proteins that insert into membranes forming ion channels. In most Cry toxins alpha-helix 2 is broken by a highly conserved proline residue (Pro70 in Cry1Ab), generating a broken-helix motif. The flexibility of the motif was altered through site-directed mutagenesis. It was found that increasing the flexibility of the motif decreased the stability, the ion transport ability and the toxicity of the protein. By removing the broken-helix motif, the biological properties were restored to a wild type level.
The interaction of carbonmonoxyhemoglobin and heme with small unilamellar phospholipid vesicles was studied using dynamic light scattering. Addition of carbonmonoxyhemoglobin to dimyristoylphosphatidylcholine:dimyristoylphosphatidylserine small unilamellar vesicles resulted in an increase of average vesicle size from 17.4 to 32.0nm. Addition of heme to vesicles produced a smaller size increase, from 17.4 to 21.0nm. Also reported is a method for preparing small unilamellar lipid vesicles of a uniform size, suitable for use in NMR spectroscopy.
Many subspecies of the soil bacterium Bacillus thuringiensis produce various parasporal crystal proteins, also known as Cry toxins, that exhibit insecticidal activity upon binding to specific receptors in the midgut of susceptible insects. One such receptor, BT-R(1) (210 kDa), is a cadherin located in the midgut epithelium of the tobacco hornworm, Manduca sexta. It has a high binding affinity (K(d) approximately 1nM) for the Cry1A toxins of B. thuringiensis. Truncation analysis of BT-R(1) revealed that the only fragment capable of binding the Cry1A toxins of B. thuringiensis was a contiguous 169-amino acid sequence adjacent to the membrane-proximal extracellular domain. The purified toxin-binding fragment acted as an antagonist to Cry1Ab toxin by blocking the binding of toxin to the tobacco hornworm midgut and inhibiting insecticidal action. Exogenous Cry1Ab toxin bound to intact COS-7 cells expressing BT-R(1) cDNA, subsequently killing the cells. Recruitment of BT-R(1) by B. thuringiensis indicates that the bacterium interacts with a specific cell adhesion molecule during its pathogenesis. Apparently, Cry toxins, like other bacterial toxins, attack epithelial barriers by targeting cell adhesion molecules within susceptible insect hosts.
Bacillus thuringiensis Cry1A toxins, in contrast to other pore-forming toxins, bind two putative receptor molecules, aminopeptidase N (APN) and cadherin-like proteins. Here we show that Cry1Ab toxin binding to these two receptors depends on the toxins' oligomeric structure. Toxin monomeric structure binds to Bt-R1, a cadherin-like protein, that induces proteolytic processing and oligomerization of the toxin (Gomez, I., Sanchez, J., Miranda, R., Bravo A., Soberon, M., FEBS Lett. (2002) 513, 242-246), while the oligomeric structure binds APN, which drives the toxin into the detergent-resistant membrane (DRM) microdomains causing pore formation. Cleavage of APN by phospholipase C prevented the location of Cry1Ab oligomer and Bt-R1 in the DRM microdomains and also attenuates toxin insertion into membranes despite the presence of Bt-R1. Immunoprecipitation experiments demonstrated that initial Cry1Ab toxin binding to Bt-R1 is followed by binding to APN. Also, immunoprecipitation of Cry1Ab toxin-binding proteins using pure oligomeric or monomeric structures showed that APN was more efficiently detected in samples immunoprecipitated with the oligomeric structure, while Bt-R1 was preferentially detected in samples immunoprecipitated with the monomeric Cry1Ab. These data agrees with the 200-fold higher apparent affinity of the oligomer than that of the monomer to an APN enriched protein extract. Our data suggest that the two receptors interact sequentially with different structural species of the toxin leading to its efficient membrane insertion.
Bacillus thuringiensis insecticidal proteins, Cry toxins, following ingestion by insect larvae, induce insecticidal effect by penetrating the brush border membranes (BBM) of midgut epithelial cells. Purified, activated B. thuringiensis Cry1Aa bound to Bombyx mori BBMV or unbound Cry1Aa were vigorously digested with Pronase. Both digests were compared by Western blotting. Free Cry1Aa was digested to alpha-helix and/or to amino acids at 1 mg Pronase/mL within 2.4 h at 37 degrees C. Whereas, BBMV-bound Cry1Aa was very resistant to Pronase digestion and even at 2 mg for 24 h, 7.5 kDa and approximately 30 kDa peptide were detected by alpha-2,3 antiserum, and alpha-4,5 and alpha-6,7 antisera, respectively. Another approximately 30 kDa peptide was also detected by beta-6-11 and domain III antisera. These fragments are believed either to be embedded in or to strongly interact with the BBMV. The 7.5 and former approximately 30 kDa peptides are thought to be derived from alpha-2,3 helix and stretch of alpha-4 to alpha-7 helices. Furthermore the latter approximately 30 kDa was thought to include the stretch of beta-6 to domain III. Moreover, the embedded Cry1Aa molecule appears to be segregated in some areas of beta-1-5 sheets, resulting in the above two approximately 30 kDa peptides. From these digestion patterns, we proposed new membrane insertion model for single Cry1Aa molecule. On the other hand, in digestion of BBMV-bound Cry1Aa, 15 kDa peptide which was recognized only by alpha-4,5 antiserum was observed. This fragment must be dimeric alpha-4,5 helices and we discussed the origin of this peptide.
The delta-endotoxin family of toxic proteins represents the major component of the insecticidal capability of the bacterium Bacillus thuringiensis. Domain I of the toxins, which is largely alpha-helical, has been proposed to unfold at protein entry into the membrane of a target insect, following models known as the penknife and umbrella models. We extended the analysis of a previous work in which four disulfide bridges were constructed in domain I of the Cry1Aa delta-endotoxin that putatively prevented unfolding during membrane partitioning. Using bioassays and voltage clamping of whole insect midgut instead of artificial lipid bilayers, it was found that, while toxicity and inhibition of the short-circuit current were reduced, only one of the disulfide bridges eliminated the activity of the toxins in the insect midgut membrane, and in that case, the loss of toxicity was due to the single amino acid substitution, R99C. It is proposed that at least alpha helices 4, 5, 6, and 7 and domain II partition in the midgut membranes of target insects, in support of an insertion model in which the whole protein translocates into the midgut membrane.