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
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
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DOI：
10.1016/j.bbapap.2016.11.011
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发表时间：
2017-02-01
影响因子：
3.2
通讯作者：
Sato, Ryoichi
中科院分区：
文献类型：
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作者：
Adegawa, Satomi;Nakama, Yui;Sato, Ryoichi
需要关于Cry毒素（由苏云金芽孢杆菌产生的杀虫蛋白）的受体相互作用区域的信息来阐明Cry毒素的作用模式并通过蛋白质工程改善其毒性。我们分析了Cry1Aa与最重要的Cry1A毒素受体之一ABC转运蛋白C2（ABCC2）的相互作用位点。竞争结合实验表明，家蚕ABCC2（BmABCC2）的Cry1A结合位点与BtR175的结合位点相同，提示Cry1Aa结构域III的环区是BtR175的结合位点。接下来，我们构建了几个结构域II环突变体毒素，并在SPR分析中测试了它们的结合亲和力，还进行了细胞肿胀试验以评估受体介导的细胞毒性。我们的研究结果表明，BtR175和BmABCC2结合所需的环区域和细胞毒性的重要区域部分重叠。我们的研究结果还表明，受体结合是必要的，但不足以细胞毒性。这是第一份报告显示ABCC2和Cry1Aa之间的相互作用区域和Cry1Aa结构域II环区域的细胞毒性相关特性。(C)2016爱思唯尔B.V.保留所有权利。
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 Ills 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. (C) 2016 Elsevier B.V. All rights reserved.