title: The domain II loops of Bacillus thuringiensis Cry1Aa form an overlapping interaction site for two Bombyx mori larvae functional receptors, ABC transporter C2 and cadherin-like receptor.
abstract: Information about the receptor-interaction region of Cry toxins, insecticidal proteins produced by Bacillus thuringiensis, is needed to elucidate the mode of action of Cry toxins and improve their toxicity through protein engineering. We analyzed the interaction sites on Cry1Aa with ABC transporter C2 (ABCC2), one of the most important Cry1A toxin receptors. A competitive binding assay revealed that the Bombyx mori ABCC2 (BmABCC2) Cry1A binding site was the same as the BtR175 binding site, suggesting that the loop region of Cry1Aa domain II is a binding site. Next, we constructed several domain II loop mutant toxins and tested their binding affinity in an SPR analysis, and also performed a cell swelling assay to evaluate receptor-mediated cytotoxicity. Our results indicate that the loop regions required for BtR175 and BmABCC2 binding and the regions important for cytotoxicity partially overlap. Our results also suggest that receptor binding is necessary but not sufficient for cytotoxicity. This is the first report showing the region of interaction between ABCC2 and Cry1Aa and the cytotoxicity-relevant properties of the Cry1Aa domain II loop region.
authors.affiliation: Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Japan.
authors.forename: Satomi
authors.initials: S
authors.lastname: Adegawa
authors.affiliation: Tokyo University of Agriculture and Technology, Japan.
authors.forename: Yui
authors.initials: Y
authors.lastname: Nakama
authors.affiliation: Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Japan; Japan Society for the Promotion of Science Research Fellowship for Young Scientists, Japan.
authors.forename: Haruka
authors.initials: H
authors.lastname: Endo
authors.affiliation: Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Shinkawa
authors.affiliation: Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Kikuta
authors.affiliation: Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Japan. Electronic address: ryoichi@cc.tuat.ac.jp.
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Sato
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000818:Animals; D001413:Bacillus thuringiensis / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D012831:Bombyx / Q000378:metabolism*; D015820:Cadherins / Q000378:metabolism*; D004731:Endotoxins / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000378:metabolism*; D019476:Insect Proteins / Q000378:metabolism; D007306:Insecticides / Q000378:metabolism; D007814:Larva / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011956:Receptors, Cell Surface / Q000378:metabolism*; D061987:Sf9 Cells
chemical_list: D018528:ATP-Binding Cassette Transporters; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D015820:Cadherins; D004731:Endotoxins; D006460:Hemolysin Proteins; D019476:Insect Proteins; D007306:Insecticides; D011956:Receptors, Cell Surface; C030956:insecticidal crystal protein, Bacillus Thuringiensis
keywords: ABC transporter C2; Bacillus thuringiensis; Cadherin-like receptor; Cry toxin; Receptor interaction site