Structure-guided design and functional characterization of an artificial red light-regulated guanylate/adenylate cyclase for optogenetic applications.
Structure-guided design and functional characterization of an artificial red light-regulated guanylate/adenylate cyclase for optogenetic applications.
复制标题
用于光遗传学应用的人造红色光调节的鸟烯酸/腺苷酸环化酶的结构引导的设计和功能表征。
DOI：
10.1074/jbc.ra118.003069
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发表时间：
2018-06-08
期刊：
影响因子：
--
通讯作者：
Winkler A
中科院分区：
文献类型：
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作者：
Etzl S;Lindner R;Nelson MD;Winkler A
光遗传学的概念是通过遗传靶向生物系统来控制光的细胞过程。尽管在这一领域取得了令人印象深刻的进展，但所采用的光感受器模块的信号转导的潜在分子机制经常没有被充分理解以合理地设计新的光遗传学工具。在这里，我们调查的要求与非天然酶效应器的红光敏感光敏色素的功能耦合，通过创建一系列的结构，具有耐辐射奇球菌细菌光敏色素连接到集胞藻鸟苷酸/腺苷酸环化酶。在我们的设计中，阐明了对环化酶调节重要的特征结构元件，我们鉴定了几种具有有希望的性质的红光调节融合体。我们提供了一个光激活的构建体的细节，该构建体具有低暗态活性和高动态范围，在体外优于以前的光遗传学工具，并扩展了我们的体内工具包，如通过操纵秀丽隐杆线虫运动活性所证明的。光敏色素连接环化酶的全长晶体结构揭示了光感受器-效应器耦合的分子细节，突出了调节环化酶元件的重要性。不同功能状态下氢氘交换的构象动力学分析丰富了我们对光敏色素信号传导和效应子信号整合的理解。我们发现，光诱导的光敏色素的构象变化不稳定的卷曲螺旋传感器效应器连接器，从抑制构象释放环化酶调节元件，增加环化酶活性的人工系统。光遗传学功能的未来设计可能受益于我们的工作，表明对效应器的合理考虑提高了初始设计的成功率，以获得具有上级特性的光遗传学工具。
Genetically targeting biological systems to control cellular processes with light is the concept of optogenetics. Despite impressive developments in this field, underlying molecular mechanisms of signal transduction of the employed photoreceptor modules are frequently not sufficiently understood to rationally design new optogenetic tools. Here, we investigate the requirements for functional coupling of red light–sensing phytochromes with non-natural enzymatic effectors by creating a series of constructs featuring the Deinococcus radiodurans bacteriophytochrome linked to a Synechocystis guanylate/adenylate cyclase. Incorporating characteristic structural elements important for cyclase regulation in our designs, we identified several red light–regulated fusions with promising properties. We provide details of one light-activated construct with low dark-state activity and high dynamic range that outperforms previous optogenetic tools in vitro and expands our in vivo toolkit, as demonstrated by manipulation of Caenorhabditis elegans locomotor activity. The full-length crystal structure of this phytochrome-linked cyclase revealed molecular details of photoreceptor–effector coupling, highlighting the importance of the regulatory cyclase element. Analysis of conformational dynamics by hydrogen–deuterium exchange in different functional states enriched our understanding of phytochrome signaling and signal integration by effectors. We found that light-induced conformational changes in the phytochrome destabilize the coiled-coil sensor–effector linker, which releases the cyclase regulatory element from an inhibited conformation, increasing cyclase activity of this artificial system. Future designs of optogenetic functionalities may benefit from our work, indicating that rational considerations for the effector improve the rate of success of initial designs to obtain optogenetic tools with superior properties.