SITE-DIRECTED MUTATIONS IN A HIGHLY CONSERVED REGION OF BACILLUS-THURINGIENSIS DELTA-ENDOTOXIN AFFECT INHIBITION OF SHORT-CIRCUIT CURRENT ACROSS BOMBYX-MORI MIDGUTS
SITE-DIRECTED MUTATIONS IN A HIGHLY CONSERVED REGION OF BACILLUS-THURINGIENSIS DELTA-ENDOTOXIN AFFECT INHIBITION OF SHORT-CIRCUIT CURRENT ACROSS BOMBYX-MORI MIDGUTS
复制标题
DOI：
10.1073/pnas.90.19.9041
复制
发表时间：
1993-10-01
影响因子：
11.1
通讯作者：
DEAN, DH
中科院分区：
文献类型：
--
作者：
CHEN, XJ;LEE, MK;DEAN, DH
苏云金芽孢杆菌δ-内毒素（Cry毒素）是以蛋白水解加工和活性形式的几乎等于65 kDa的杀虫蛋白。这些毒素之一CryIIIA的结构已由Li等人确定[Li，J.，卡罗尔，J.& Ellar，D. J.（1991）Nature（伦敦）353，815-821]，并且包含三个结构域。据信，其他δ-内毒素采用类似的三维结构。Li等人提出第一个结构域是膜孔形成结构域。我们实验室以前的工作表明，第二个结构域是受体结合结构域，但第三个结构域的功能尚不清楚。定点诱变用于将一个高度保守的区域（CryIAa的QRYRVRIRYAS（残基525-535））的“精氨酸面”转化为选择的其它残基。该序列对应于第三结构域中CryIIIA的β-折叠17。第二和第三精氨酸位置的突变导致蛋白质的结构改变，并且在大肠杆菌中表达不佳。从基因突变，以取代赖氨酸的第一和第四个毒素的表达和结构不变，胰蛋白酶活化，CD光谱，和受体结合，但大大减少了其杀虫特性和抑制短路电流在家蚕中肠。有人提出，该区域发挥了作用，毒素功能的离子通道。
Bacillus thuringiensis delta-endotoxins (Cry toxins) are insecticidal proteins of almost-equal-to 65 kDa in the proteolytically processed and active form. The structure of one of these toxins, CryIIIA, has been determined by Li et al. [Li, J., Carroll, J. & Ellar, D. J. (1991) Nature (London) 353, 815-821] and contains three domains. It is believed that other delta-endotoxins adopt similar three-dimensional structure. Li et al. proposed that the first domain is the membrane pore-forming domain. Previous work from our laboratory has shown that the second domain is the receptor binding domain, but the function of the third domain is unclear. Site-directed mutagenesis was used to convert the ''arginine face'' of one of rive highly conserved regions, QRYRVRIRYAS of CryIAa (residues 525-535), to selected other residues. This sequence corresponds to the beta-sheet 17 of CryIIIA in the third domain. Mutations in the second and third arginine positions resulted in structural alterations in the protein and were poorly expressed in Escherichia coli. Toxins from genes mutated to replace lysine for the first and fourth arginines were unaltered in expression and structure, as measured by trypsin activation, CD spectra, and receptor binding, but were substantially reduced in their insecticidal properties and inhibition of short circuit current across Bombyx mori midguts. It is proposed that this region plays a role in toxin function as an ion channel.