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针对杀蚊Mpp46Ab毒素跨膜β-发夹结构中K残基的随机突变分析

Random Mutational Analysis Targeting Residue K within the Transmembrane β-Hairpin of the Mosquitocidal Mpp46Ab Toxin.

作者信息

Miyazaki Midoka, Asakura Mami, Ide Toru, Hayakawa Tohru

机构信息

Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan.

出版信息

Biology (Basel). 2023 Dec 1;12(12):1481. doi: 10.3390/biology12121481.

Abstract

Mpp46Ab is a mosquito-larvicidal pore-forming toxin derived from TK-E6. Pore formation is believed to be a central mode of Mpp46Ab action, and the cation selectivity of the channel pores, in particular, is closely related to its mosquito-larvicidal activity. In the present study, we constructed a mutant library in which residue K within the transmembrane β-hairpin was randomly replaced with other amino acid residues. Upon mutagenesis and following primary screening using mosquito larvae, we obtained 15 mutants in addition to the wild-type toxin. Bioassays using purified proteins revealed that two mutants, K155E and K155I, exhibited toxicity significantly higher than that of the wild-type toxin. Although increased cation selectivity was previously reported for K155E channel pores, we demonstrated in the present study that the cation selectivity of K155I channel pores was also significantly increased. Considering the characteristics of the amino acids, the charge of residue 155 may not directly affect the cation selectivity of Mpp46Ab channel pores. Replacement of K with glutamic acid or isoleucine may induce a similar conformational change in the region associated with the ion selectivity of the Mpp46Ab channel pores. Mutagenesis targeting the transmembrane β-hairpin may be an effective strategy for enhancing the ion permeability of the channel pores and the resulting mosquito-larvicidal activity of Mpp46Ab.

摘要

Mpp46Ab是一种源自TK-E6的杀蚊幼虫成孔毒素。成孔被认为是Mpp46Ab作用的核心模式,尤其是通道孔的阳离子选择性与其杀蚊幼虫活性密切相关。在本研究中,我们构建了一个突变体文库,其中跨膜β-发夹内的K残基被其他氨基酸残基随机取代。诱变后,使用蚊幼虫进行初步筛选,除野生型毒素外,我们还获得了15个突变体。使用纯化蛋白进行的生物测定表明,两个突变体K155E和K155I表现出明显高于野生型毒素的毒性。尽管之前报道K155E通道孔的阳离子选择性增加,但我们在本研究中证明K155I通道孔的阳离子选择性也显著增加。考虑到氨基酸的特性,155位残基的电荷可能不会直接影响Mpp46Ab通道孔的阳离子选择性。用谷氨酸或异亮氨酸取代K可能会在与Mpp46Ab通道孔离子选择性相关的区域诱导类似的构象变化。针对跨膜β-发夹的诱变可能是增强通道孔离子通透性以及由此提高Mpp46Ab杀蚊幼虫活性的有效策略。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ba17/10741074/26ae5a05d759/biology-12-01481-g001.jpg

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