Structure-based protein engineering for α-amylase inhibitory activity of plant defensin
Structure-based protein engineering for α-amylase inhibitory activity of plant defensin
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DOI：
10.1002/prot.21378
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发表时间：
2007-08-01
影响因子：
2.9
通讯作者：
Lyu, Ping-Chiang
中科院分区：
文献类型：
--
作者：
Lin, Ku-Feng;Lee, Tian-Ren;Lyu, Ping-Chiang
用H-1核磁共振波谱法测定了从绿豆种子中分离到的一种新的植物防御素的结构。VrD 2（V radiant plant defensin 2蛋白）的三维结构包括由四个二硫键稳定的α-螺旋和一个三链反平行β-折叠。这种蛋白质既不显示杀虫活性，也不显示α-淀粉酶抑制活性，尽管显示出与VrD 1相似的全局折叠，VrD 1是一种杀虫植物防御素，已被认为通过抑制昆虫α-淀粉酶而起作用。我们以前的研究提出，植物防御素的L3环是重要的这种抑制。结构分析和表面电荷比较VrD 1和VrD 2显示，L3的带电残基与观察到的这些蛋白质的抑制活性的差异相关。通过将提议的VrD 1功能环转移到VrD 2的结构等效环上而产生的VrD 2嵌合体支持这一假设。VrD 2嵌合体对黄粉虫α-淀粉酶具有明显的抑制活性，与VrD 2嵌合体相比，VrD 2嵌合体仅相差5个残基。这些结果阐明了α-淀粉酶抑制植物防御素的模式，也代表了一种可能的方法，为工程新的α-淀粉酶抑制剂。植物防御素是植物天然免疫系统的重要组成部分，因此蛋白质工程的应用将为植物防御提供一种有效的方法。
The structure of a novel plant defensin isolated from the seeds of the mung bean, Vigna radiate, has been determined by H-1 nuclear magnetic resonance spectroscopy. The three-dimensional structure of VrD2, the V radiate plant defensin 2 protein, comprises an alpha-helix and one triplestranded anti-parallel beta-sheet stabilized by four disulfide bonds. This protein exhibits neither insecticidal activity nor alpha-amylase inhibitory activity in spite of showing a similar global fold to that of VrD1, an insecticidal plant defensin that has been suggested to function by inhibiting insect alpha-amylase. Our previous study proposed that loop L3 of plant defensins is important for this inhibition. Structural analyses and surface charge comparisons of VrD1 and VrD2 revealed that the charged residues of L3 correlate with the observed difference in inhibitory activities of these proteins. A VrD2 chimera that was produced by transferring the proposed functional loop of VrD1 onto the structurally equivalent loop of VrD2 supported this hypothesis. The VrD2 chimera, which differs by only five residues compared with VrD2, showed obvious activity against Tenebrio molitor alpha-amylase. These results clarify the mode of alpha-amylase inhibition of plant defensins and also represent a possible approach for engineering novel alpha-amylase inhibitors. Plant defensins are important constituents of the innate immune system of plants, and thus the application of protein engineering to this protein family may provide an efficient method for protecting against crop losses.