Toxins (Basel). 2020 Feb 22;12(2):137. doi: 10.3390/toxins12020137.
Destruxin A (DA), a cyclodepsipeptidic mycotoxin produced by entomopathogenic fungus , has good insecticidal activity and potential to be a new pesticide. However, the mechanism of action is still obscure. Our previous experiments showed that DA was involved in regulation of transcription and protein synthesis and suggested that silkworms' arginine tRNA synthetase (BmArgRS), Lamin-C Proteins (BmLamin-C) and ATP-dependent RNA helicase PRP1 (BmPRP1) were candidates of DA-binding proteins. In this study, we employed bio-layer interferometry (BLI), circular dichroism (CD), cellular thermal shift assay (CETSA), and other technologies to verify the interaction of DA with above three proteins in vitro and in vivo. The results of BLI indicated that BmArgRS and BmLamin-C were binding-protein of DA with K value 5.53 × 10 and 8.64 × 10 M, but not BmPRP1. These interactions were also verified by CD and CETSA tests. In addition, docking model and mutants assay in vitro showed that BmArgRS interacts with DA at the pocket including Lys228, His231, Asp434 and Gln437 in its enzyme active catalysis region, while BmLamin-C binds to DA at His524 and Lys528 in the tail domain. This study might provide new insight and evidence in illustrating molecular mechanism of DA in breaking insect.
杀虫素 A（DA）是一种由昆虫病原真菌产生的环二肽类真菌毒素，具有良好的杀虫活性和成为新型农药的潜力。然而，其作用机制尚不清楚。我们之前的实验表明，DA 参与转录和蛋白质合成的调节，提示家蚕的精氨酸 tRNA 合成酶（BmArgRS）、核纤层蛋白（BmLamin-C）和 ATP 依赖的 RNA 解旋酶 PRP1（BmPRP1）可能是 DA 结合蛋白的候选者。在这项研究中，我们采用生物层干涉（BLI）、圆二色性（CD）、细胞热转移分析（CETSA）和其他技术，在体外和体内验证了 DA 与上述三种蛋白质的相互作用。BLI 的结果表明，BmArgRS 和 BmLamin-C 是 DA 的结合蛋白，其 K 值分别为 5.53×10 和 8.64×10 M，但 BmPRP1 不是。这些相互作用也通过 CD 和 CETSA 测试得到了验证。此外，体外对接模型和突变体实验表明，BmArgRS 在其酶活性催化区域的口袋中与 DA 相互作用，口袋包括 Lys228、His231、Asp434 和 Gln437，而 BmLamin-C 在尾部结构域中与 DA 结合在 His524 和 Lys528 上。本研究可能为阐明 DA 破坏昆虫的分子机制提供新的见解和证据。
