The determination of the receptor‐binding region of Cry toxins produced by Bacillus thuringiensis is expected to facilitate an improvement in their insecticidal ability through protein engineering. We analyzed the region on Cry1Aa molecules involved in interactions with the cadherin‐like protein receptor BtR175 using cysteine‐substituted mutant toxins and several synthetic peptides corresponding to the loops in domain 2. In addition, the region necessary to trigger oligomerization was analyzed using these mutant toxins. The mutant toxins were modified by two types of molecule, i.e. digested fragments of the Cry1Aa precursor with an average molecular mass of 2 kDa and 5‐iodoacetamidofluorescein, which has a molecular mass of 515 kDa. We examined whether these modifications interfere with the toxin–BtR175 interaction as a result of steric hindrance. 5‐Iodoacetamidofluorescein modification of R311C, N376C and G442C revealed steric hindrance effects, indicating that R311 on loop 1, N376 on loop 2 and G442 on loop 3 are on the contact face of the toxin–BtR175 interface when Cry1Aa binds to BtR175. Loop 2 is thought to interact with BtR175 directly, as a peptide corresponding to the N‐terminal half of loop 2, (365)LYRRIILG(372), has the potential to bind to BtR175 fragments. Meanwhile, mutant toxins with cysteine substitutions in loops 1 and 2 were oligomerized by the binding of digested fragments in the activation process without receptor interaction, and the wild‐type toxin formed oligomers by interaction with BtR175 fragments. These observations suggest that loops 1 and 2 form both a binding region and a sensor region, which triggers toxin oligomer formation. Structured digital abstract http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7259673, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7259722, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7259737, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7259757, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7259774, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7259791, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7259808, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7259685, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7259707, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7259830: btr175 (uniprotkb:http://www.ebi.uniprot.org/entry/Q9XY09) binds (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) to cry1Aa (uniprotkb:http://www.ebi.uniprot.org/entry/P0A366) by surface plasmon resonance (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0107)

To identify and gain a better understanding of the cadherin‐like receptor‐binding site on Bacillus thuringiensis Cry toxins, it is advantageous to use Cry1Aa toxin, because its 3D structure is known. Therefore, Cry1Aa toxin was used to examine the locations of cadherin‐like protein‐binding sites. Initial experiments examining the binding compatibility for Cry1Aa toxin of partial fragments of recombinant proteins of a 175 kDa cadherin‐like protein from Bombyx mori (BtR175) and another putative receptor for Cry1Aa toxin, aminopeptidase N1, from Bo. mori (BmAPN1), suggested that their binding sites are close to each other. Of the seven mAbs against Cry1Aa toxin, two mAbs were selected that block the binding site for BtR175 on Cry1Aa toxin: 2A11 and 2F9. Immunoblotting and alignment analyses of four Cry toxins revealed amino acids that included the epitope of mAb 2A11, and suggested that the area on Cry1Aa toxin blocked by the binding of mAb 2A11 is located in the region consisting of loops 2 and 3. Two Cry1Aa toxin mutants were constructed by substituting a Cys on the area blocked by the binding of mAb 2A11, and the small blocking molecule, N‐(9‐acridinyl)maleimide, was introduced at each Cys substitution to determine the BtR175‐binding site. Substitution of Tyr445 for Cys had a crippling effect on binding of Cry1Aa toxin to BtR175, suggesting that Tyr445 may be in or close to the BtR175‐binding site. Monoclonal antibodies that blocked the binding site for BtR175 on Cry1Aa toxin inhibited the toxicity of Cry1Aa toxin against Bo. mori, indicating that binding of Cry1Aa toxin to BtR175 is essential for the action of Cry1Aa toxin on the insect.