Morse R J, Yamamoto T, Stroud R M
Department of Biochemistry and Biophysics, University of California, San Francisco, 94143, San Francisco, CA, USA.
Structure. 2001 May 9;9(5):409-17. doi: 10.1016/s0969-2126(01)00601-3.
Genetically modified (GM) crops that express insecticidal protein toxins are an integral part of modern agriculture. Proteins produced by Bacillus thuringiensis (Bt) during sporulation mediate the pathogenicity of Bt toward a spectrum of insect larvae whose breadth depends upon the Bt strain. These transmembrane channel-forming toxins are stored in Bt as crystalline inclusions called Cry proteins. These proteins are the active agents used in the majority of biorational pesticides and insect-resistant transgenic crops. Though Bt toxins are promising as a crop protection alternative and are ecologically friendlier than synthetic organic pesticides, resistance to Bt toxins by insects is recognized as a potential limitation to their application.
We have determined the 2.2 A crystal structure of the Cry2Aa protoxin by multiple isomorphous replacement. This is the first crystal structure of a Cry toxin specific to Diptera (mosquitoes and flies) and the first structure of a Cry toxin with high activity against larvae from two insect orders, Lepidoptera (moths and butterflies) and Diptera. Cry2Aa also provides the first structure of the proregion of a Cry toxin that is cleaved to generate the membrane-active toxin in the larval gut.
The crystal structure of Cry2Aa reported here, together with chimeric-scanning and domain-swapping mutagenesis, defines the putative receptor binding epitope on the toxin and so may allow for alteration of specificity to combat resistance or to minimize collateral effects on nontarget species. The putative receptor binding epitope of Cry2Aa identified in this study differs from that inferred from previous structural studies of other Cry toxins.
表达杀虫蛋白毒素的转基因作物是现代农业的一个重要组成部分。苏云金芽孢杆菌（Bt）在芽孢形成过程中产生的蛋白质介导了Bt对一系列昆虫幼虫的致病性，其作用范围取决于Bt菌株。这些跨膜通道形成毒素在Bt中以称为Cry蛋白的晶体包涵体形式储存。这些蛋白质是大多数生物合理农药和抗虫转基因作物中使用的活性剂。尽管Bt毒素有望成为一种作物保护替代品，并且比合成有机农药对生态更友好，但昆虫对Bt毒素的抗性被认为是其应用的一个潜在限制。
我们通过多同晶置换法确定了Cry2Aa原毒素的2.2埃晶体结构。这是第一个针对双翅目（蚊子和苍蝇）的Cry毒素晶体结构，也是第一个对鳞翅目（蛾和蝴蝶）和双翅目昆虫幼虫具有高活性的Cry毒素结构。Cry2Aa还提供了Cry毒素前区的第一个结构，该前区在幼虫肠道中被切割以产生膜活性毒素。
本文报道的Cry2Aa晶体结构，连同嵌合扫描和结构域交换诱变，定义了毒素上假定的受体结合表位，因此可能允许改变特异性以对抗抗性或最小化对非靶标物种的附带影响。本研究中鉴定的Cry2Aa假定受体结合表位与先前对其他Cry毒素的结构研究推断的表位不同。
