Protein engineering of Bacillus thuringiensis delta-endotoxin: Mutations at domain II of CryIAb enhance receptor affinity and toxicity toward gypsy moth larvae
Protein engineering of Bacillus thuringiensis delta-endotoxin: Mutations at domain II of CryIAb enhance receptor affinity and toxicity toward gypsy moth larvae
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DOI：
10.1073/pnas.93.25.14338
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发表时间：
1996-12-10
影响因子：
11.1
通讯作者：
Dean, DH
中科院分区：
文献类型：
--
作者：
Rajamohan, F;Alzate, O;Dean, DH
利用定点突变技术构建了苏云金芽孢杆菌δ-内毒素CryIAb结构域II环残基的取代或缺失，以研究其在受体结合和对舞毒蛾（Lymantria dispar）的毒性中的功能作用。用Ala或Gly（N372 A，N372 G）取代环2残基N372使对舞毒蛾幼虫的毒性增加8倍，并且使对舞毒蛾中肠刷状缘膜囊泡（BBMV）的结合亲和力增加约4倍。然而，N372（D3）的缺失显著降低了毒性（>21倍）以及结合亲和力，表明残基N372参与受体结合。有趣的是，与野生型毒素相比，三重突变体DF-1（N372 A、A282 G和L283 S）对舞毒蛾新生儿的毒性增加了36倍。DF-1的活性增强与较高的结合亲和力（18倍）和结合位点浓度相关。解离结合测定表明，BBMV结合的突变体毒素的解离速率与野生型相似。然而，DF-1毒素的结合比野生型和N372 A毒素多4倍。并且其与结合亲和力和效力直接相关。用标记的N372 A，DF-1和CryIAb毒素探测的舞毒蛾BBMV的蛋白质印迹识别了一种常见的210-kDa蛋白，表明突变体的活性增加不是由与其他受体结合引起的。N372 A和DF-1的结合亲和力的改善表明这些环处的较短侧链可能是。这些结果为通过改善受体结合相互作用来工程化具有更高效力和更宽靶标害虫谱的δ-内毒素提供了极好的模型系统。
Substitutions or deletions of domain II loop residues of Bacillus thuringiensis delta-endotoxin CryIAb were constructed using site-directed mutagenesis techniques to investigate their functional roles in receptor binding and toxicity toward gypsy moth (Lymantria dispar). Substitution of loop 2 residue N372 with Ala or Gly (N372A, N372G) increased the toxicity against gypsy moth larvae 8-fold and enhanced binding affinity to gypsy moth midgut brush border membrane vesicles (BBMV) approximate to 4-fold. Deletion of N372 (D3), however, substantially reduced toxicity (>21 times) as well as binding affinity suggesting that residue N372 is involved in receptor binding, Interestingly, a triple mutant, DF-1 (N372A, A282G and L283S), has a 36-fold increase in toxicity to gypsy moth neonates compared with wild-type toxin. The enhanced activity of DF-1 was correlated with higher binding affinity (18-fold) and binding site concentrations, Dissociation binding assays suggested that the off-rate of the BBMV-bound mutant toxins was similar to that of the wild type. However, DF-1 toxin bound 4 times more than the wild-type and N372A toxins. and it was directly correlated with binding affinity and potency. Protein blots of gypsy moth BBMV probed with labeled N372A, DF-1, and CryIAb toxins recognized a common 210-kDa protein, indicating that the increased activity of the mutants was not caused by binding to additional receptor(s). The improved binding affinity of N372A and DF-1 suggest that a shorter side chain at these loops ma). fit the toxin more efficiently to the binding pockets, These results offer an excellent model system for engineering delta-endotoxins with higher potency and wider spectra of target pests by improving receptor binding interactions.