Directly light-regulated binding of RGS-LOV photoreceptors to anionic membrane phospholipids.
Directly light-regulated binding of RGS-LOV photoreceptors to anionic membrane phospholipids.
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DOI：
10.1073/pnas.1802832115
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发表时间：
2018-08-14
影响因子：
11.1
通讯作者：
Chow BY
中科院分区：
文献类型：
--
作者：
Glantz ST;Berlew EE;Jaber Z;Schuster BS;Gardner KH;Chow BY
光-氧-电压(LOV)域光感受器广泛存在于自然界，具有多种不同的信号作用和机制。在这里，我们展示了一类真菌LOV蛋白通过蓝光开关静电相互作用与阴离子质膜磷脂动态结合。这种可逆的联系由蓝光迅速触发，当照明停止时，这种联系在几秒钟内停止。在天然宿主中，我们预测这些蛋白通过控制G蛋白信号转导区域的融合调节因子(RGS)结构域的招募来调节G蛋白信号；在应用环境中，我们预计这些蛋白的工程嵌合版本将有助于通过与膜本身的直接相互作用来快速光遗传膜定位融合蛋白，而不需要额外的组件来指导亚细胞定位。我们报道了具有蓝光开关、高亲和力(KD∼10−7M)的自然光-氧-电压(LOV)光感受器，以及与阴离子磷脂的直接静电相互作用。在酵母和哺乳动物细胞表达系统中，通过生物信息学、计算蛋白质建模、结构功能研究和光遗传学分析揭示，灰霉病菌的光受体BcLOV4的膜定位直接与其黄素光循环相连，并由LOV传感器与其未知功能的C末端结构域(DUF)之间的连接区的多碱基两亲性螺旋介导。在模型系统中，BcLOV4迅速从胞浆转移到质膜(∼1秒)。可逆的静电相互作用在阴离子磷脂中是非选择性的，表现出结合强度取决于膜的总阴离子含量，而不优先于特定的头基。体外和细胞反应也观察到了BcLOV4同源物，因此很可能是普遍的Dikarya LOV类，其成员与G蛋白信号转导(RGS)结构域相关。目前尚不知道天然光感受器以光依赖的方式直接与膜磷脂结合，因此本工作建立了一种光传感信号传输模式和一种单组分光遗传工具，该工具具有快速的膜定位动力学，接近扩散极限。
Light–oxygen–voltage (LOV) domain photoreceptors are found ubiquitously in nature and possess highly diverse signaling roles and mechanisms. Here, we show that a class of fungal LOV proteins dynamically associates with anionic plasma membrane phospholipids by a blue light-switched electrostatic interaction. This reversible association is rapidly triggered by blue light and ceases within seconds when illumination ceases. Within the native host, we predict that these proteins regulate G-protein signaling by the controlled recruitment of fused regulator of G-protein signaling (RGS) domains; in applied contexts, we anticipate that engineered chimeric versions of such proteins will be useful for rapid optogenetic membrane localization of fused proteins through direct interaction with the membrane itself, without requiring additional components to direct subcellular localization. We report natural light–oxygen–voltage (LOV) photoreceptors with a blue light-switched, high-affinity (KD ∼ 10−7 M), and direct electrostatic interaction with anionic phospholipids. Membrane localization of one such photoreceptor, BcLOV4 from Botrytis cinerea, is directly coupled to its flavin photocycle, and is mediated by a polybasic amphipathic helix in the linker region between the LOV sensor and its C-terminal domain of unknown function (DUF), as revealed through a combination of bioinformatics, computational protein modeling, structure–function studies, and optogenetic assays in yeast and mammalian cell line expression systems. In model systems, BcLOV4 rapidly translocates from the cytosol to plasma membrane (∼1 second). The reversible electrostatic interaction is nonselective among anionic phospholipids, exhibiting binding strengths dependent on the total anionic content of the membrane without preference for a specific headgroup. The in vitro and cellular responses were also observed with a BcLOV4 homolog and thus are likely to be general across the dikarya LOV class, whose members are associated with regulator of G-protein signaling (RGS) domains. Natural photoreceptors are not previously known to directly associate with membrane phospholipids in a light-dependent manner, and thus this work establishes both a photosensory signal transmission mode and a single-component optogenetic tool with rapid membrane localization kinetics that approaches the diffusion limit.
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