Composition of the Putative Prepore Complex of Bacillus thuringiensis Cry1Ab Toxin.

Prepore formation is hypothesized to be an obligate step in the insertion of Cry1Ab toxin into insect brush border membrane vesicles. We examined the architecture of the putative prepore when isolated using the published protocols [1] [2]. Our results demonstrate that the putative prepore form of Cry1Ab is a combination of receptor proteins attached to the toxin, when purified. The results also suggest that this prepore form as prepared by the methods published is different from other membrane-extracted oligomeric forms of Cry toxins and prepore of other toxins in general. While most other known prepores are composed of multimers of a single protein, the Cry1Ab prepore, as generated, is a protein-receptor complex oligomer and monomers of Cry toxins.

Cry protein crystals: a novel platform for protein delivery.

Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium Bacillus thuringiensis. We demonstrate that fusion of the cry3Aa gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications. These Cry3Aa fusion protein crystals are efficiently taken up and retained by macrophages and other cell lines in vitro, and can be delivered to mice in vivo via multiple modes of administration. Oral delivery of Cry3Aa fusion protein crystals to C57BL/6 mice leads to their uptake by MHC class II cells, including macrophages in the Peyer's patches, supporting the notion that the Cry3Aa framework can be used to stabilize cargo protein against degradation for delivery to gastrointestinal lymphoid tissues.

Characterization of a Mutant Bacillus thuringiensis d-endotoxin with enhanced stability and toxicity / Caracterización de una delta endotoxina mutante de Bacillus thuringiensis con estabilidad y toxicidad aumentadas

The centrally located a-helix 5 of Bacillus thuringiensis d-endotoxins is critical for insect toxicity through ion-channel formation. We analyzed the role of the highly conserved residue Histidine 168 (H168) using molecular biology, electrophysiology and biophysical techniques. Toxin H168R was ~3-fold more toxic than the wild type (wt) protein whereas H168Q was 3 times less toxic against Manduca sexta. Spectroscopic analysis revealed that the H168Q and H168R mutations did not produce gross structural alterations, and that H168R (Tm= 59 °C) was more stable than H168Q (Tm= 57.5 °C) or than the wt (Tm= 56 °C) toxins. These three toxins had similar binding affinities for larval midgut vesicles (Kcom) suggesting that the differences in toxicity did not result from changes in initial receptor binding. Dissociation binding assays and voltage clamping analysis suggest that the reduced toxicity of the H168Q toxin may result from reduced insertion and/or ion channel formation. In contrast, the H168R toxin had a greater inhibition of the short circuit current than the wt toxin and an increased rate of irreversible binding (kobs), consistent with its lower LC50 value. Molecular modeling analysis suggested that both the H168Q and H168R toxins could form additional hydrogen bonds that could account for their greater thermal stability. In addition to this, it is likely that H168R has an extra positive charge exposed to the surface which could increase its rate of insertion into susceptible membranes.

La a-Hélice 5 del domino I de las d-endotoxinas de Bacillus thuringiensis, es crítica para la toxicidad de las toxinas contra insectos al participar en la formación de canales iónicos. La participación en la función tóxica del residuo Histidina 168 (H168) –el cual es altamente conservado– fue estudiada mediante técnicas de biología molecular, electrofisiología y biofísica. La toxina mutante H168R fue ~ 3 veces más tóxica que la toxina silvestre (ts) en Manduca sexta, mientras que H168Q fue 3 veces menos tóxica. Los análisis espectroscópicos indicaron que las mutaciones no producen alteraciones estructurales significativas y que la toxina H168R (Tm= 59 °C) es más estable que las toxinas H168Q (Tm= 57.5 °C) y wt (Tm= 56 °C). Las tres toxinas exhibieron uniones de afinidad similares (Kcom) en vesículas de intestino de larvas de insecto, indicando que las diferencias en la toxicidad no se deben a cambios en la unión inicial al receptor. Los ensayos de unión/disociación y fijación de voltaje mostraron que la reducción de la toxicidad de la toxina H168Q se puede atribuir a una disminución en la inserción y/o en la formación de canales iónicos. De otro lado, H168R mostró una inhibición a la corriente de corto circuito mayor que la ts y un aumento en unión irreversible (kobs), lo cual es consistente con un menor valor de CL50. La modelación molecular sugiere que H168Q y H168R forman puentes de hidrógeno adicionales, lo que les confiere mayor estabilidad térmica. Adicionalmente, es probable que H168R tenga una carga positiva extra expuesta en la superficie, lo cual aumentaría su tasa de inserción en membranas susceptibles.

Bacillus thuringiensis Cry2Ab is active on Anopheles mosquitoes: single D block exchanges reveal critical residues involved in activity.

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.

Preferential protection of domains II and III of bacillus thuringiensis cry1Aa toxin by brush border membrane vesicles / Protección preferencial de los dominios II y III de la toxina cry1Aa de bacillus thuringiensis en vesículas de membrana de borde de cepillo

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.

La superficie de la toxina Cry1Aa, en el asa 2 del dominio II contiene expuesta la leucina 371, la cual fue modificada a lisina produciendo una mutante sensible a la tripsina, L371K. Esta mutante produce dos fragmentos de 37 y 26 kDa por acción de la tripsina que son separables por SDS-PAGE, pero que a la purificación por cromatografía líquida se mantienen como una sola molécula de 65 kDa. El fragmento grande contiene al dominio I y una parte del dominio II (aminoácidos 1 al 371). El polipéptido de 26 kDa contiene la parte restante del dominio II y dominio III (aminoácidos 372 al 609). Cuando la toxina mutante fue tratada con dosis altas de jugo intestinal de Manduca sexta, ambos fragmentos fueron degradados. Sin embargo, cuando fueron incubados en VMBC de M. sexta, el fragmento de 26 kDa fue protegido preferencialmente de las proteasas intestinales. Como se ha reportado, la toxina silvestre Cry1Aa también es protegida de la degradación de las proteasas cuando es incubada en VMBC de M. sexta. Sin embargo, cuando se adicionó VMBC de ratón a la mezcla de reacción, ni la toxina Cry1Aa ni la mutante L371K mostraron resistencia a las proteasas y fueron degradadas. 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.

Participation of valine 171 in alpha-Helix 5 of Bacillus thuringiensis Cry1Ab delta-endotoxin in translocation of toxin into Lymantria dispar midgut membranes.

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.

Ser170 of Bacillus thuringiensis Cry1Ab delta-endotoxin becomes anchored in a hydrophobic moiety upon insertion of this protein into Manduca sexta brush border membranes.

BACKGROUND: Three spin-labeled mutant proteins, mutated at the beginning, middle, and end of alpha-helix 5 of the Bacillus thuringiensis Cry1Ab delta-endotoxin, were used to study the involvement of these specific amino acid residues in ion transport and to determine conformational changes in the vicinity of these residues when the protein was translocated into a biological membrane. RESULTS: Amino acid residue leucine 157, located in the N-terminal portion of alpha-helix 5, showed no involvement in ion transport, and the environment that surrounds the residue did not show any change when transferred into the biological membrane. Serine 170, located in the middle of the alpha-helix, showed no involvement in ion transport, but our findings indicate that in the membrane-bound state this residue faces an environment that makes the spin less mobile, as opposed to the mobility observed in an aqueous environment. Serine 176, located in the C-terminal end of the alpha-helix 5 is shown to be involved in ion transport activity. CONCLUSION: Ion transport data for L157, S170, and S176, along with the mobility of the spin-labels, structural characterization of the resulting proteins, and toxicity assays against a target insect, suggest that the toxin undergoes conformational changes upon protein translocation into the midgut membrane. These conformational changes result in the midregion of the alpha-helix 5 being exposed to a hydrophobic-like environment. The location of these three residues in the toxin suggests that the entire alpha-helix becomes inserted in the insect midgut membrane.

Transgenic rice plants expressing a modified cry1Ca1 gene are resistant to Spodoptera litura and Chilo suppressalis.

Nucleotide sequence encoding the truncated insecticidal Cry1Ca1 protein from Bacillus thuringiensis was extensively modified based on the codon usage of rice genes. The overall G + C contents of the synthetic cry1Ca1 coding sequence were raised to 65% with an additional bias of enriching for G and C ending codons as preferred by monocots. The synthetic gene was introduced into the Chinese japonica variety, Xiushui 11, by Agrobacterium-mediated transformation. Transgenic rice plants harboring this gene were highly resistant to Chilo suppressalis and Spodoptera litura larvae as revealed by insect bioassays. High levels of Cry1Ca1 protein were obtained in the leaves of transgenic rice, which were effective in achieving 100% mortality of S. litura and C. suppressalis larvae. The levels of Cry1Ca1 expression in the leaves of these transgenic plants were up to 0.34% of the total soluble proteins. The larvae of C. suppressalis and S. litura could consume a maximum of 1.89 and 4.89 mm2 of transgenic leaf area whereas the consumption of nontransgenic leaves by these larvae was significantly higher; 58.33 and 61.22 mm2, respectively. Analysis of R1 transgenic plants indicated that the cry1Ca1 was inherited by the progeny plants and provided complete protection against C. suppressalis and S. litura larvae.

Mutagenic analysis of putative domain II and surface residues in mosquitocidal Bacillus thuringiensis Cry19Aa toxin.

The mosquitocidal crystal protein, Cry19Aa, from Bacillus thuringiensis ssp. jegathesan, has high toxicity to Anopheles stephensi and Culex pipiens but is less toxic to Aedes aegypti. To study the functional role of putative domain II and surface residues in mosquito toxicity, 16 alanine substitution mutations were introduced into Cry19Aa. All mutant constructs were expressed as 65-kDa protoxins and subsequently digested by trypsin to produce further fragmented polypeptides of 40 and 25 kDa. With chymotrypsin, however, most protoxins were digested to 60 kDa and minor bands. The circular dichroism spectra of the chymotrypsin-activated toxins of Cry19Aa and muteins, Y324A, W357A, Y412A, Y414A, W416A, D418A and F485A indicated that there was no significant variation in their structure. In mosquito bioassays, Y324A, W357A, Y410A, W416A, D418A and F485A muteins showed substantial reductions in mosquitocidal activity toward A. aegypti and C. pipiens. These muteins also showed reduced competition with wild-type fluorescein 5-isothiocyanate-labeled Cry19Aa for binding to C. pipiens brush border membrane vesicles. These data suggest that the reduction of toxicity was a result of the reduced binding affinity. From these studies we have identified loop residues of domain II that are important in toxicity and receptor binding to Culex larval midgut.

Proteomic analysis of the mosquito Aedes aegypti midgut brush border membrane vesicles.

We analyzed brush border membrane vesicle proteins from isolated midguts of the mosquito Aedes aegypti, by two proteomic methods: two-dimensional gel electrophoresis (isoelectric focusing and SDS-PAGE) and a shotgun two-dimensional liquid chromatographic (LS/LS) approach based on multidimensional protein identification technology (MudPIT). We were interested in the most abundant proteins of the apical brush border midgut membrane. About 400 spots were detected on 2D gels and 39 spots were cored and identified by mass spectrometry. 86 proteins were identified by MudPIT. Three proteins, arginine kinase, putative allergen and actin are shown to be the most predominant proteins in the sample. The total number of 36 proteins detected by both methods represents the most abundant proteins in the BBMV.

All domains of Cry1A toxins insert into insect brush border membranes.

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 alpha-helix 5 of domain I along with adjacent helices alpha-4 or alpha-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 alpha-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.

Membrane insertion of the Bacillus thuringiensis Cry1Ab toxin: single mutation in domain II block partitioning of the toxin into the brush border membrane.

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.

Blocking binding of Bacillus thuringiensis Cry1Aa to Bombyx mori cadherin receptor results in only a minor reduction of toxicity.

BACKGROUND: Bacillus thuringiensis Cry1Aa insecticidal protein is the most active known B. thuringiensis toxin against the forest insect pest Lymantria dispar (gypsy moth), unfortunately it is also highly toxic against the non-target insect Bombyx mori (silk worm). RESULTS: Surface exposed hydrophobic residues over domains II and III were targeted for site-directed mutagenesis. Substitution of a phenylalanine residue (F328) by alanine reduced binding to the Bombyx mori cadherin by 23-fold, reduced biological activity against B. mori by 4-fold, while retaining activity against Lymantria dispar. CONCLUSION: The results identify a novel receptor-binding epitope and demonstrate that virtual elimination of binding to cadherin BR-175 does not completely remove toxicity in the case of B. mori.

Identification of a Bacillus thuringiensis Cry11Ba toxin-binding aminopeptidase from the mosquito, Anopheles quadrimaculatus.

BACKGROUND: Aminopeptidase N (APN) type proteins isolated from several species of lepidopteran insects have been implicated as Bacillus thuringiensis (Bt) toxin-binding proteins (receptors) for Cry toxins. We examined brush border membrane vesicle (BBMV) proteins from the mosquito Anopheles quadrimaculatus to determine if APNs from this organism would bind mosquitocidal Cry toxins that are active to it. RESULTS: A 100-kDa protein with APN activity (APNAnq 100) was isolated from the brush border membrane of Anopheles quadrimaculatus. Native state binding analysis by surface plasmon resonance shows that APNAnq 100 forms tight binding to a mosquitocidal Bt toxin, Cry11Ba, but not to Cry2Aa, Cry4Ba or Cry11Aa. CONCLUSION: An aminopeptidase from Anopheles quadrimaculatus mosquitoes is a specific binding protein for Bacillus thuringiensis Cry11Ba.

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.

Redesigning Bacillus thuringiensis Cry1Aa toxin into a mosquito toxin.

The Bacillus thuringiensis crystal protein Cry1Aa is normally selectively active to caterpillar larvae. Through rational design, toxicity (microg/ml) to the mosquito Culex pipiens was introduced by selected deletions and substitutions of the loop residues of domain II. Toxicity to its natural target Manduca sexta was concomitantly abolished. The successful grafting of the alternate mosquito toxicity onto the original lepidopteran Cry1Aa toxin demonstrates the possibility of designing and engineering a desired toxicity into any toxin of a common scaffold by reshaping the receptor binding region with desired specificities.

Structural implication of the induced immune response by Bacillus thuringiensis Cry proteins: role of the N-terminal region.

The potential role of the regions (carboxil and amino) of the Cry proteins in the ability of these proteins to elicit strong immune responses was investigated. Intraperitoneal immunization of mice with the homologous Cry1A protoxins (130-133 kDa), with the long C-terminal half gave rise mostly to similar, strong serum and mucosal IgG and IgM antibody response but a lower induction of these Ab by intranasal route. Remarkably, Cry3A protoxin, devoid of C-terminal half was able to induce a significant mucosal IgG, and IgM Ab as well as Cry1A protoxins, suggesting us that immunogenic abilities are not restricted to C-terminal half but N-terminal half itself could be involved. In fact, this assumption was strengthen by the strong immunogenic abilities of the Cry1A toxins, specially IgG and IgA Ab induced by both routes in different mucosal sites. These data indicate that immunogenic abilities of the Bt Cry proteins reside and depends of the N-terminal half.

Enhancement of Cry19Aa mosquitocidal activity against Aedes aegypti by mutations in the putative loop regions of domain II.

Improvements in the mosquitocidal activity of Bacillus thuringiensis Cry19Aa were achieved by protein engineering of putative surface loop residues in domain II through rational design. The improvement of Aedes toxicity in Cry19Aa was 42,000-fold and did not affect its toxicity against Anopheles or Culex.

Bacillus thuringiensis delta-endotoxin binding to brush border membrane vesicles of rice stem borers.

The receptor binding step in the molecular mode of action of five delta-endotoxins (Cry1Ab, Cry1Ac, Cry1C, Cry2A, and Cry9C) from Bacillus thuringiensis was examined to find toxins with different receptor sites in the midgut of the striped stem borer (SSB) Chilo suppressalis (Walker) and yellow stem borer (YSB) Scirpophaga incertulas (Walker) (Lepidoptera: Pyralidae). Homologous competition assays were used to estimate binding affinities (K(com)) of (125)I-labelled toxins to brush border membrane vesicles (BBMV). The SSB BBMV affinities in decreasing order was: Cry1Ab = Cry1Ac > Cry9C > Cry2A > Cry1C. In YSB, the order of decreasing affinities was: Cry1Ac > Cry1Ab > Cry9C = Cry2A > Cry1C. The number of binding sites (B(max)) estimated by homologous competition binding among the Cry toxins did not affect toxin binding affinity (K(com)) to both insect midgut BBMVs. Results of the heterologous competition binding assays suggest that Cry1Ab and Cry1Ac compete for the same binding sites in SSB and YSB. Other toxins bind with weak (Cry1C, Cry2A) or no affinity (Cry9C) to Cry1Ab and Cry1Ac binding sites in both species. Cry2A had the lowest toxicity to 10-day-old SSB and Cry1Ab and Cry1Ac were the most toxic. Taken together, the results of this study show that Cry1Ab or Cry1Ac could be combined with either Cry1C, Cry2A, or Cry9C for more durable resistance in transgenic rice. Cry1Ab should not be used together with Cry1Ac because a mutation in one receptor site could diminish binding of both toxins.

Molecular basis for Bacillus thuringiensis Cry1Ab toxin specificity: two structural determinants in the Manduca sexta Bt-R1 receptor interact with loops alpha-8 and 2 in domain II of Cy1Ab toxin.

The identification of epitopes involved in Cry toxin-receptor interaction could provide insights into the molecular basis of insect specificity and for designing new toxins to overcome the potential problem of insect resistance. In previous works, we determined that the Manduca sexta Cry1A cadherin-like receptor (Bt-R(1)) interacts with Cry1A toxins through epitope (865)NITIHITDTNN(875) and by loop 2 of domain II in the toxin (Gomez, I., Miranda-Rios, J., Rudiño-Piñera, E., Oltean, D. I., Gill, S. S., Bravo, A., and Soberón, M. (2002) J. Biol. Chem. 277, 30137-30143.). In this work, we narrowed to 12 amino acids a previously identified Bt-R(1) 66 amino acids epitope (Dorsch, J. A., Candas, M., Griko, N. B., Maaty, W. S. A., Midbo, E. G., Vadlamudi, R. K., and Bulla, L. A., Jr. (2002) Insect Biochem. Mol. Biol. 32, 1025-1036) and identified loop alpha-8 of Cry1Ab domain II as its cognate binding epitope. Two amino acid Bt-R(1) toxin binding regions of 70 residues, one comprised of residues 831-900 containing the (865)NITIHITDTNN(875) epitope (TBR1) and the other comprised of residues 1291-1360 (TBR2) were cloned by RT-PCR and produced in Escherichia coli. Cry1A toxins bind with the two TBR regions in contrast with the nontoxic Cry3A toxin. The loop 2 synthetic peptide competed with the binding of Cry1Ab toxin to both TBR regions in contrast to the alpha-8 synthetic peptide that only competed with Cry1Ab binding to TBR2. Western blots and competition ELISA analysis showed that the Cry1Ab loop 2 RR368-9EE mutant did not show observable binding to TBR1 but still bound the TBR2 peptide. This result suggests that loop alpha-8 interacts with the TBR2 region. Competition ELISA analysis of Cry1Ab binding to the two TBR peptides revealed that the toxin binds the TBR1 region with 6-fold higher affinity than the TBR2 region. The amino acid sequence of TBR2 involved on Cry1Ab interaction was narrowed to 12 amino acids, (1331)IPLPASILTVTV(1342), by using synthetic peptides as competitors for Cry1Ab binding to Bt-R(1). Our results show that the specificity of Cry1A involves at least two structural determinants on both molecules.