Engineering proteins for allosteric control by light or ligands
Engineering proteins for allosteric control by light or ligands
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DOI：
10.1038/s41596-019-0165-3
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发表时间：
2019-06-01
期刊：
影响因子：
14.8
通讯作者：
Hahn, Klaus M.
中科院分区：
文献类型：
--
作者：
Dagliyan, Onur;Dokholyan, Nikolay, V;Hahn, Klaus M.
控制活细胞中的蛋白质活性可以揭示时空动力学在信号通路中的作用。具有工程化变构反应的蛋白质类似物在蛋白质信号传导的询问中可以是特别有效的，因为它们可以以最小的天然相互作用扰动来取代内源性蛋白质。然而，鉴定靶蛋白中的变构位点一直是一个挑战，其中响应结构域的插入产生与天然蛋白质的活性相当的变构反应。在这里，我们描述了一种详细的方案来产生可以通过雷帕霉素或蓝光变构控制的蛋白质的遗传编码类似物，以及在体外和哺乳动物细胞系中产生和测试这些类似物的实验方法。我们描述了计算方法，晶体结构或同源模型的基础上，以确定插入工程雷帕霉素响应（uniRapR）域或光响应光氧电压2（LOV 2）域的有效位点。插入的结构域变构调节活性位点，以不可逆的激活响应雷帕霉素，或以更高的空间和时间分辨率可逆失活响应光。这些策略已成功应用于蛋白激酶、Rho家族GTP酶和鸟嘌呤交换因子（GEF）的催化结构域，以及GEF Vav 2的结合结构域。计算任务可以在几个小时内完成，然后进行1-2周的实验验证。我们提供的工程蛋白的计算设计，克隆和实验测试的协议，使用Src酪氨酸激酶，GEF Vav 2，和Rho GTdR Rac 1作为例子。
Control of protein activity in living cells can reveal the role of spatiotemporal dynamics in signaling circuits. Protein analogs with engineered allosteric responses can be particularly effective in the interrogation of protein signaling, as they can replace endogenous proteins with minimal perturbation of native interactions. However, it has been a challenge to identify allosteric sites in target proteins where insertion of responsive domains produces an allosteric response comparable to the activity of native proteins, Here, we describe a detailed protocol to generate genetically encoded analogs of proteins that can be allosterically controlled by either rapamycin or blue light, as well as experimental procedures to produce and test these analogs in vitro and in mammalian cell lines. We describe computational methods, based on crystal structures or homology models, to identify effective sites for insertion of either an engineered rapamycin-responsive (uniRapR) domain or the light-responsive light-oxygen-voltage 2 (LOV2) domain. The inserted domains allosterically regulate the active site, responding to rapamycin with irreversible activation, or to light with reversible inactivation at higher spatial and temporal resolution. These strategies have been successfully applied to catalytic domains of protein kinases, Rho family GTPases, and guanine exchange factors (GEFs), as well as the binding domain of a GEF Vav2. Computational tasks can be completed within a few hours, followed by 1-2 weeks of experimental validation. We provide protocols for computational design, cloning, and experimental testing of the engineered proteins, using Src tyrosine kinase, GEF Vav2, and Rho GTPase Rac1 as examples.