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The determination of the receptor-binding region of Cry toxins produced byBacillus thuringiensisis 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 syn-thetic peptides corresponding to the loops in domain 2. | ỊFEBS Journal Analysis of the region for receptor binding and triggering of oligomerization on Bacillus thuringiensis CrylAa toxin Fumiaki Obata Madoka Kitami Yukino Inoue Shogo Atsumi Yasutaka Yoshizawa and Ryoichi Sato Graduate Schoolof Bio-Applications and Systems Engineering Tokyo University of Agriculture and Technology Japan Keywords Bacillus thuringiensis Bombyx mori BtR175 Cry1Aa oligomerization Correspondence R. Sato Graduate Schoolof Bio-Applications and Systems Engineering Tokyo University of Agriculture and Technology Koganei Tokyo 184-8588 Japan Fax 81 42 388 7277 Tel 81 42 388 7277 E-mail ryoichi@cc.tuat.ac.jp Received 4 June 2009 revised 3 August 2009 accepted 12 August 2009 doi 10.1111 j.1742-4658.2009.07275.x 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-Iodoacetami-dofluorescein 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 CrylAa 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 .
