Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins
Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins
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DOI：
10.1073/pnas.1417910112
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发表时间：
2015-01-06
影响因子：
11.1
通讯作者：
Kuhlman, Brian
中科院分区：
文献类型：
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作者：
Guntas, Gurkan;Hallett, Ryan A.;Kuhlman, Brian
光诱导的蛋白质-蛋白质相互作用的发现允许对各种生物过程进行空间和时间控制。为了有效，光二聚化剂应该具有几个特征：它应该在光刺激时显示出结合亲和力的大变化，它不应该与细胞中的其他分子交叉反应，并且它应该容易用于各种生物体中以募集彼此感兴趣的蛋白质。为了创建符合这些标准的开关，我们将细菌SsrA肽嵌入天然存在的光开关的C-末端螺旋中，即来自燕麦的光-氧-电压2（LOV 2）结构域。在黑暗中，SsrA肽在空间上被阻止结合其天然结合配偶体SspB。当用蓝光激活时，LOV 2结构域的C-末端螺旋从蛋白质中脱离，允许SsrA肽结合SspB。在没有优化的情况下，开关在光刺激下对SspB的结合亲和力表现出两倍的变化。在这里，我们描述了使用计算蛋白质设计，噬菌体展示，和高通量结合试验，以创建一个改进的光诱导二聚体（iLID），改变其亲和力SspB超过50倍的光刺激。iLID的晶体结构显示了LOV 2结构域的表面与苯丙氨酸之间的关键相互作用，苯丙氨酸被工程化以在黑暗中更紧密地将SsrA肽固定在LOV 2结构域上。我们通过在哺乳动物细胞培养中的光介导的亚细胞定位和小GT3信号的可逆控制证明了开关的功能效用。
The discovery of light-inducible protein-protein interactions has allowed for the spatial and temporal control of a variety of biological processes. To be effective, a photodimerizer should have several characteristics: it should show a large change in binding affinity upon light stimulation, it should not cross-react with other molecules in the cell, and it should be easily used in a variety of organisms to recruit proteins of interest to each other. To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photo-switch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa. In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB. Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation. Here, we describe the use of computational protein design, phage display, and high-throughput binding assays to create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation. A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark. We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.