Nanobody-enabled monitoring of kappa opioid receptor states
Nanobody-enabled monitoring of kappa opioid receptor states
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DOI：
10.1038/s41467-020-14889-7
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发表时间：
2020-03-02
影响因子：
16.6
通讯作者：
Roth, Bryan L.
中科院分区：
文献类型：
--
作者：
Che, Tao;English, Justin;Roth, Bryan L.
最近的研究表明，尽管由于缺乏合适的工具，我们对gpcr进行查询、监测和可视化的能力受到限制，但gpcr在多种状态之间迅速相互转换。我们之前报道了两种纳米体（Nb39和Nb6），它们稳定了kappa阿片受体不同的配体和药效分隔的构象。在这里，我们通过x射线晶体学证明了一个纳米体靶向的变构结合位点，Nb6通过该位点稳定了依赖于配体的非活性状态。当Nb39稳定活性状态时，我们展示了这两种状态依赖的纳米体如何在细胞原位提供配体稳定状态的实时报告。值得注意的是，我们证明嵌合gpcr可以用工程纳米体结合位点创建，以报告配体稳定状态。我们的研究结果提供了关于纳米体变构调节KOR的潜在机制的见解，以及报告GPCR活性实时、原位动态范围的工具。近年来的研究表明，G蛋白偶联受体在多种状态之间快速相互转换。在这里，作者使用kappa阿片受体（KOR），并展示了两种状态依赖的纳米体如何通过KOR和其他gpcr提供配体稳定状态的实时报告。
Recent studies show that GPCRs rapidly interconvert between multiple states although our ability to interrogate, monitor and visualize them is limited by a relative lack of suitable tools. We previously reported two nanobodies (Nb39 and Nb6) that stabilize distinct ligand- and efficacy-delimited conformations of the kappa opioid receptor. Here, we demonstrate via X-ray crystallography a nanobody-targeted allosteric binding site by which Nb6 stabilizes a ligand-dependent inactive state. As Nb39 stabilizes an active-like state, we show how these two state-dependent nanobodies can provide real-time reporting of ligand stabilized states in cells in situ. Significantly, we demonstrate that chimeric GPCRs can be created with engineered nanobody binding sites to report ligand-stabilized states. Our results provide both insights regarding potential mechanisms for allosterically modulating KOR with nanobodies and a tool for reporting the real-time, in situ dynamic range of GPCR activity.Recent studies revealed that G protein-coupled receptors rapidly interconvert between multiple states. Here, authors use the kappa opioid receptor (KOR) and show how two state-dependent nanobodies provide real-time reporting of ligand stabilized states with KOR and other GPCRs.