Tryptophan spectroscopy studies and black lipid bilayer analysis indicate that the oligomeric structure of Cry1Ab toxin from Bacillus thuringiensis is the membrane-insertion intermediate

During intoxication, the Cry protoxins must change from insoluble crystals into membrane-inserted toxins, which form ionic pores. Binding of Cry1A toxins to the cadherin receptor promotes the formation of a 250 kDa oligomer. In this work, we analyzed for the first time the structural changes presented by Cry1Ab toxin upon membrane insertion. Trp fluorescence of pure monomeric and oligomeric structures in solution and in a membrane-bound state was analyzed. Cry1Ab has nine Trp residues, seven of them in pore-forming domain I. Trp quenching analysis with iodide indicated that oligomerization caused a 27% reduction in the level of Trp exposed to the solvent. Most of the oligomeric structure (96%) inserts into the membrane as a function of the lipid:protein ratio, in contrast to the monomer (10%). Additionally, the membrane-associated oligomer presented a blue shift of 5 nm in lambda(max) of the emission spectrum, indicating a more hydrophobic environment for some Trp residues. In agreement with this, iodide was unable to quench the Trp of the membrane-bound oligomer, suggesting that a significant part of the protein may be buried in the membrane. Quenching analysis using brominated and spin-labeled phospholipids in the vesicles indicates that most of the Trp residues are located close to the membrane-water interface. Finally, ionic currents in black lipid bilayers revealed that the oligomeric structure has kinetics different from those of the monomer, producing stable channels with a high probability of being open in contrast to the monomer that exhibited unstable opening patterns. These data show that the oligomer, in contrast to the monomer, is able to interact efficiently with phospholipid membranes forming stable pores.     

Unfolding events in the water-soluble monomeric Cry1Ab toxin during transition to oligomeric pre-pore and membrane-inserted pore channel

The insecticidal crystal (Cry) proteins produced by Bacillus thuringiensis undergo several conformational changes from crystal inclusion protoxins to membrane-inserted channels in the midgut epithelial cells of the target insect. Here we analyzed the stability of the different forms of Cry1Ab toxin, monomeric toxin, pre-pore complex, and membrane-inserted channel, after urea and thermal denaturation by monitoring intrinsic tryptophan fluorescence of the protein and 1-anilinonaphthalene-8-sulfonic acid binding to partially unfolded proteins. Our results showed that flexibility of the monomeric toxin was dramatically enhanced upon oligomerization and was even further increased by insertion of the pre-pore into the membrane as shown by the lower concentration of chaotropic agents needed to achieve unfolding of the oligomeric species. The flexibility of the toxin structures is further increased by alkaline pH. We found that the monomer-monomer interaction in the pre-pore is highly stable because urea promotes oligomer denaturation without disassembly. Partial unfolding and limited proteolysis studies demonstrated that domains II and III were less stable and unfold first, followed by unfolding of the most stable domain I, and also that domain I is involved in monomer-monomer interaction. The thermal-induced unfolding and analysis of energy transfer from Trp residues to bound 1-anilinonaphthalene-8-sulfonic acid dye showed that in the membrane-inserted pore domains II and III are particularly sensitive to heat denaturation, in contrast to domain I, suggesting that only domain I may be inserted into the membrane. Finally, the insertion into the membrane of the oligomeric pre-pore structure was not affected by pH. However, a looser conformation of the membrane-inserted domain I induced by neutral or alkaline pH correlates with active channel formation. Our studies suggest for the first time that a more flexible conformation of Cry toxin could be necessary for membrane insertion, and this flexible structure is induced by toxin oligomerization. Finally the alkaline pH found in the midgut lumen of lepidopteran insects could increase the flexibility of membrane-inserted domain I necessary for pore formation.     

Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains

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 (Gómez, I., Sánchez, J., Miranda, R., Bravo A., Soberón, 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.     

Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains

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.     

Cytotoxicity of Bacillus thuringiensis Cry1Ab toxin depends on specific binding of the toxin to the cadherin receptor BT-R1 expressed in insect cells

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.     

Domains II and III of Bacillus thuringiensis Cry1Ab toxin remain exposed to the solvent after insertion of part of domain I into the membrane

Bacillus thuringiensis produces insecticidal proteins named Cry toxins, that are used commercially for the control of economical important insect pests. These are pore-forming toxins that interact with different receptors in the insect gut, forming pores in the apical membrane causing cell burst and insect death. Elucidation of the structure of the membrane-inserted toxin is important to fully understand its mechanism of action. One hypothesis proposed that the hairpin of α-helices 4-5 of domain I inserts into the phospholipid bilayer, whereas the rest of helices of domain I are spread on the membrane surface in an umbrella-like conformation. However, a second hypothesis proposed that the three domains of the Cry toxin insert into the bilayer without major conformational changes. In this work we constructed single Cys Cry1Ab mutants that remain active against Manduca sexta larvae and labeled them with different fluorescent probes that have different responses to solvent polarity. Different soluble quenchers as well as a membrane-bound quencher were used to compare the properties of the soluble and brush border membrane-inserted forms of Cry1Ab toxin. The fluorescence and quenching analysis presented here, revealed that domains II and III of the toxin remain in the surface of the membrane and only a discrete region of domain I is inserted into the lipid bilayer, supporting the umbrella model of toxin insertion.     

Structural changes of the Cry1Ac oligomeric pre-pore from bacillus thuringiensis induced by N-acetylgalactosamine facilitates toxin membrane insertion

The primary action of Cry toxins produced by Bacillus thuringiensis is to lyse midgut epithelial cells in their target insect by forming lytic pores. The toxin-receptor interaction is a complex process, involving multiple interactions with different receptor and carbohydrate molecules. It has been proposed that Cry1A toxins sequentially interact with a cadherin receptor, leading to the formation of a pre-pore oligomer structure, and that the oligomeric structure binds to glycosylphosphatidyl-inositol-anchored aminopeptidase-N (APN) receptor. The Cry1Ac toxin specifically recognizes the N-acetylgalactosamine (GalNAc) carbohydrate present in the APN receptor from Manduca sexta larvae. In this work, we show that the Cry1Ac pre-pore oligomer has a higher binding affinity with APN than the monomeric toxin. The effects of GalNAc binding on the toxin structure were studied in the monomeric Cry1Ac, in the soluble pre-pore oligomeric structure, and in its membrane inserted state by recording the fluorescence status of the tryptophan (W) residues. Our results indicate that the W residues of Cry1Ac have a different exposure to the solvent when compared with that of the closely related Cry1Ab toxin. GalNAc binding specifically affects the exposure of W545 in the pre-pore oligomer in contrast to the monomer where GalNAc binding did not affect the fluorescence of the toxin. These results indicate a subtle conformational change in the GalNAc binding pocket in the pre-pore oligomer that could explain the increased binding affinity of the Cry1Ac pre-pore to APN. Although our analysis did not reveal major structural changes in the pore-forming domain I upon GalNAc binding, it showed that sugar interaction enhanced membrane insertion of soluble pre-pore oligomeric structure. Therefore, the data presented here permits to propose a model in which the interaction of Cry1Ac pre-pore oligomer with APN receptor facilitates membrane insertion and pore formation.     

A trimeric building block model for Cry toxins in vitro ion channel formation

The crystal (Cry) insecticidal toxins, or δ-endotoxins, are lethal to a wide variety of insect larvae, and are therefore very important in insect control. Toxicity has been explained by formation of transmembrane oligomeric pores or ion channels and, more recently, by the ability of the monomeric toxin to subvert cellular signaling pathways. The structure, topology, and precise role of the putative pore in toxicity are not known. However, in vitro biophysical studies suggest that helices α4 and α5 in domain I insert into the lipid bilayer as an α-helical hairpin. Mutagenesis studies have assigned an important role to α5 in maintaining oligomerization, and to α4 in channel formation. To detect the possible homo-oligomerizing tendencies of these two helices, we have used the evolutionary conservation data contained in sixteen Cry homologs in order to filter non-native interactions found during a global conformational search. No conserved homo-oligomer was found for α4, but a right handed trimeric α5 model was present in the simulations of all Cry sequences. We propose a model for Cry toxin oligomerization based on sequence analysis and available mutagenesis data.     

Bacillus thuringiensis Cry1Ab mutants affecting oligomer formation are non-toxic to Manduca sexta larvae

Pore-forming toxins are biological weapons produced by a variety of living organisms, particularly bacteria but also by insects, reptiles, and invertebrates. These proteins affect the cell membrane of their target, disrupting permeability and leading eventually to cell death. The pore-forming toxins typically transform from soluble, monomeric proteins to oligomers that form transmembrane channels. The Cry toxins produced by Bacillus thuringiensis are widely used as insecticides. These proteins have been recognized as pore-forming toxins, and their primary action is to lyse midgut epithelial cells in their target insect. To exert their toxic effect, a prepore oligomeric intermediate is formed leading finally to membrane-inserted oligomeric pores. To understand the role of Cry oligomeric pre-pore formation in the insecticidal activity we isolated point mutations that affected toxin oligomerization but not their binding with the cadherin-like, Bt-R(1) receptor. We show the helix alpha-3 in domain I contains sequences that could form coiled-coil structures important for oligomerization. Some single point mutants in this helix bound Bt-R(1) receptors with similar affinity as the wild-type toxin, but were affected in oligomerization and were severally impaired in pore formation and toxicity against Manduca sexta larvae. These data indicate the pre-pore oligomer and the toxin pore formation play a major role in the intoxication process of Cry1Ab toxin in insect larvae.     

Characterization of the mechanism of action of the genetically modified Cry1AbMod toxin that is active against Cry1Ab-resistant insects

Bacillus thuringiensis Cry toxins are used in the control of insect pests. They are pore-forming toxins with a complex mechanism that involves the sequential interaction with receptors. They are produced as protoxins, which are activated by midgut proteases. Activated toxin binds to cadherin receptor, inducing an extra cleavage including helix α-1, facilitating the formation of a pre-pore oligomer. The toxin oligomer binds to secondary receptors such as aminopeptidase and inserts into lipid rafts forming pores and causing larval death. The primary threat to efficacy of Bt-toxins is the evolution of insect resistance. Engineered Cry1AMod toxins, devoid of helix α-1, could be used for the control of resistance in lepidopterans by bypassing the altered cadherin receptor, killing resistant insects affected in this receptor. Here we analyzed the mechanism of action of Cry1AbMod. We found that alkaline pH and the presence of membrane lipids facilitates the oligomerization of Cry1AbMod. In addition, tryptophan fluorescence emission spectra, ELISA binding to pure aminopeptidase receptor, calcein release assay and analysis of ionic-conductance in planar lipid bilayers, indicated that the secondary steps in mode of action that take place after interaction with cadherin receptor such as oligomerization, receptor binding and pore formation are similar in the Cry1AbMod and in the wild type Cry1Ab. Finally, the membrane-associated structure of Cry1AbMod oligomer was analyzed by electron crystallography showing that it forms a complex with a trimeric organization.     

The pre-pore from Bacillus thuringiensis Cry1Ab toxin is necessary to induce insect death in Manduca sexta

The insecticidal Cry toxins from Bacillus thuringiensis bacteria are pore-forming toxins that lyse midgut epithelial cells in insects. We have previously proposed that they form pre-pore oligomeric intermediates before membrane insertion. For formation of these oligomers coiled-coil structures are important, and helix alpha-3 from Cry toxins could form coiled-coils. Our data shows that different mutations in helix alpha-3 are affected in pore formation and toxicity. Mutants affected in toxicity bind Bt-R(1) receptor with a similar K(D) as the wild type toxin but do not form oligomers nor induce pore formation in planar lipid bilayers, indicating that the pre-pore oligomer is an obligate intermediate in the intoxication of Cry1Ab toxin and that interaction of monomeric Cry1Ab with Bt-R(1) is not enough to kill susceptible larvae.     

Role of tryptophan residues in toxicity of Cry1Ab toxin from Bacillus thuringiensis

Bacillus thuringiensis produces insecticidal proteins (Cry protoxins) during the sporulation phase as parasporal crystals. During intoxication, the Cry protoxins must change from insoluble crystals into membrane-inserted toxins which form ionic pores. The structural changes of Cry toxins during oligomerization and insertion into the membrane are still unknown. The Cry1Ab toxin has nine tryptophan residues; seven are located in domain I, the pore-forming domain, and two are located in domain II, which is involved in receptor recognition. Eight Trp residues are highly conserved within the whole family of three-domain Cry proteins, suggesting an essential role for these residues in the structural folding and function of the toxin. In this work, we analyzed the role of Trp residues in the structure and function of Cry1Ab toxin. We replaced the Trp residues with phenylalanine or cysteine using site-directed mutagenesis. Our results show that W65 and W316 are important for insecticidal activity of the toxin since their replacement by Phe reduced the toxicity against Manduca sexta. The presence of hydrophobic residue is important at positions 117, 219, 226, and 455 since replacement by Cys affected either the crystal formation or the insecticidal activity of the toxin in contrast to replacement by Phe in these positions. Additionally, some mutants in positions 219, 316, and 455 were also affected in binding to brush border membrane vesicles (BBMV). This is the first report that studies the role of Trp residues in the activity of Cry toxins.     

A trimeric building block model for Cry toxins in vitro ion channel formation

The crystal (Cry) insecticidal toxins, or delta-endotoxins, are lethal to a wide variety of insect larvae, and are therefore very important in insect control. Toxicity has been explained by formation of transmembrane oligomeric pores or ion channels and, more recently, by the ability of the monomeric toxin to subvert cellular signaling pathways. The structure, topology, and precise role of the putative pore in toxicity are not known. However, in vitro biophysical studies suggest that helices alpha4 and alpha5 in domain I insert into the lipid bilayer as an alpha-helical hairpin. Mutagenesis studies have assigned an important role to alpha5 in maintaining oligomerization, and to alpha4 in channel formation. To detect the possible homo-oligomerizing tendencies of these two helices, we have used the evolutionary conservation data contained in sixteen Cry homologs in order to filter non-native interactions found during a global conformational search. No conserved homo-oligomer was found for alpha4, but a right handed trimeric alpha5 model was present in the simulations of all Cry sequences. We propose a model for Cry toxin oligomerization based on sequence analysis and available mutagenesis data.     

Role of Tryptophan Residues in Toxicity of Cry1Ab Toxin from Bacillus thuringiensis

Bacillus thuringiensis produces insecticidal proteins (Cry protoxins) during the sporulation phase as parasporal crystals. During intoxication, the Cry protoxins must change from insoluble crystals into membrane-inserted toxins which form ionic pores. The structural changes of Cry toxins during oligomerization and insertion into the membrane are still unknown. The Cry1Ab toxin has nine tryptophan residues; seven are located in domain I, the pore-forming domain, and two are located in domain II, which is involved in receptor recognition. Eight Trp residues are highly conserved within the whole family of three-domain Cry proteins, suggesting an essential role for these residues in the structural folding and function of the toxin. In this work, we analyzed the role of Trp residues in the structure and function of Cry1Ab toxin. We replaced the Trp residues with phenylalanine or cysteine using site-directed mutagenesis. Our results show that W65 and W316 are important for insecticidal activity of the toxin since their replacement by Phe reduced the toxicity against Manduca sexta. The presence of hydrophobic residue is important at positions 117, 219, 226, and 455 since replacement by Cys affected either the crystal formation or the insecticidal activity of the toxin in contrast to replacement by Phe in these positions. Additionally, some mutants in positions 219, 316, and 455 were also affected in binding to brush border membrane vesicles (BBMV). This is the first report that studies the role of Trp residues in the activity of Cry toxins.     

Role of receptor interaction in the mode of action of insecticidal Cry and Cyt toxins produced by Bacillus thuringiensis

Cry toxins from Bacillus thuringiensis are used for insect control. Their primary action is to lyse midgut epithelial cells. In this review we will summarize recent findings on the Cry toxin-receptor interaction and the role of receptor recognition in their mode of action. Cry toxins interact sequentially with multiple receptors. In lepidopteran insects, Cry1A monomeric toxins interact with the first receptor and this interaction triggers oligomerization of the toxins. The oligomer then interacts with second receptor inducing insertion into membrane microdomains and larval death. In the case of mosquitocidal toxins, Cry and Cyt toxins play a part. These toxins have a synergistic effect and Cyt1Aa overcomes Cry toxin resistance. Recently, it was proposed that Cyt1Aa synergizes or suppresses resistance to Cry toxins by functioning as a membrane-bound receptor for Cry toxin.     

Trapping of Vibrio cholerae Cytolysin in the Membrane-bound Monomeric State Blocks Membrane Insertion and Functional Pore Formation by the Toxin

Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging cytolytic toxin that belongs to the family of β barrel pore-forming protein toxins. VCC induces lysis of its target eukaryotic cells by forming transmembrane oligomeric β barrel pores. The mechanism of membrane pore formation by VCC follows the overall scheme of the archetypical β barrel pore-forming protein toxin mode of action, in which the water-soluble monomeric form of the toxin first binds to the target cell membrane, then assembles into a prepore oligomeric intermediate, and finally converts into the functional transmembrane oligomeric β barrel pore. However, there exists a vast knowledge gap in our understanding regarding the intricate details of the membrane pore formation process employed by VCC. In particular, the membrane oligomerization and membrane insertion steps of the process have only been described to a limited extent. In this study, we determined the key residues in VCC that are critical to trigger membrane oligomerization of the toxin. Alteration of such key residues traps the toxin in its membrane-bound monomeric state and abrogates subsequent oligomerization, membrane insertion, and functional transmembrane pore-formation events. The results obtained from our study also suggest that the membrane insertion of VCC depends critically on the oligomerization process and that it cannot be initiated in the membrane-bound monomeric form of the toxin. In sum, our study, for the first time, dissects membrane binding from the subsequent oligomerization and membrane insertion steps and, thus, defines the exact sequence of events in the membrane pore formation process by VCC.     

Structural and functional studies of alpha-helix 5 region from Bacillus thuringiensis Cry1Ab delta-endotoxin

The crystal insecticidal proteins from Bacillus thuringiensis are modular proteins comprised of three domains connected by single linkers. Domain I is a seven alpha-helix bundle, which has been involved in membrane insertion and pore formation activity. Site-directed mutagenesis has contributed to identify regions that might play an important role in the structure of the pore-forming domain within the membrane. There are several evidences that support that the hairpin alpha4-alpha5 inserts into the membrane in an antiparallel manner, while other helices lie on the membrane surface. We hypothesized that highly conserved residues of alpha5 could play an important role in toxin insertion, oligomerization and/or pore formation. A total of 15 Cry1Ab mutants located in six conserved residues of Cry1Ab, Y153, Y161, H168, R173, W182 and G183, were isolated. Eleven mutants were located within helix alpha5, one mutant was located in the loop alpha4-alpha5 and three mutants, W182P, W182I and G183C, were located in the loop alpha5-alpha6. Their effect on binding, K(+) permeability and toxicity against Manduca sexta larvae was analyzed and compared. The results provide direct evidence that some residues located within alpha5 have an important role in stability of the toxin within the insect gut, while some others also have an important role in pore formation. The results also provide evidence that conserved residues within helix alpha5 are not involved in oligomer formation since mutations in these residues are able to make pores in vitro.     

Identification of Aminopeptidase-N2 as a Cry2Ab binding protein in Manduca sexta

Bacillus thuringiensis Cry2Ab toxin has been used in combination with Cry1Ac for resistance management on the Bt-cotton that is widely planted worldwide. However, little is known regarding Cry2Ab mode of action. Particularly, there is a gap of knowledge on the identification of insect midgut proteins that bind Cry2Ab and mediate toxicity. In the case of Cry1Ab toxin, a transmembrane cadherin protein and glycosyl-phosphatidylinositol (GPI) anchored proteins like aminopeptidase-N1 (APN1) or alkaline-phosphatase (ALP) from Manduca sexta, have been shown to be important for oligomer formation and insertion into the membrane. Binding competition experiments showed that Cry2Ab toxin does not share binding sites with Cry1Ab toxin in M. sexta brush border membrane vesicles (BBMV). Also, that Cry2Ab shows reduced binding to the Cry1Ab binding molecules cadherin, APN1 or ALP. Finally, ligand blot experiments and protein sequence by LC–MS/MS identified APN2 isoform as a Cry2Ab binding protein. Cloning and expression of APN2 confirmed that APN2 is a Cry2Ab binding protein.     

Domain II Loop 3 of Bacillus thuringiensis Cry1Ab Toxin Is Involved in a ��Ping Pong�� Binding Mechanism with Manduca sexta Aminopeptidase-N and Cadherin Receptors

Bacillus thuringiensis Cry toxins are used worldwide as insecticides in agriculture, in forestry, and in the control of disease transmission vectors. In the lepidopteran Manduca sexta, cadherin (Bt-R₁) and aminopeptidase-N (APN) function as Cry1A toxin receptors. The interaction with Bt-R₁ promotes cleavage of the amino-terminal end, including helix α-1 and formation of prepore oligomer that binds to APN, leading to membrane insertion and pore formation. Loops of domain II of Cry1Ab toxin are involved in receptor interaction. Here we show that Cry1Ab mutants located in domain II loop 3 are affected in binding to both receptors and toxicity against Manduca sexta larvae. Interaction with both receptors depends on the oligomeric state of the toxin. Monomers of loop 3 mutants were affected in binding to APN and to a cadherin fragment corresponding to cadherin repeat 12 but not with a fragment comprising cadherin repeats 7–12. In contrast, the oligomers of loop 3 mutants were affected in binding to both Bt-R₁ fragments but not to APN. Toxicity assays showed that either monomeric or oligomeric structures of Cry1Ab loop 3 mutations were severely affected in insecticidal activity. These data suggest that loop 3 is differentially involved in the binding with both receptor molecules, depending on the oligomeric state of the toxin and also that possibly a “ping pong” binding mechanism with both receptors is involved in toxin action.