Development of "Plug and Play'' Fiducial Marks for Structural Studies of GPCR Signaling Complexes by Single-Particle Cryo-EM
Development of "Plug and Play'' Fiducial Marks for Structural Studies of GPCR Signaling Complexes by Single-Particle Cryo-EM
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DOI：
10.1016/j.str.2019.10.004
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发表时间：
2019-12-03
期刊：
影响因子：
5.7
通讯作者：
Kossiakoff, Anthony A.
中科院分区：
文献类型：
--
作者：
Dutka, Przemyslaw;Mukherjee, Somnath;Kossiakoff, Anthony A.
通过定制噬菌体展示选择生成基于“通用”合成抗体（sAB）的基准标记，以方便使用单粒子冷冻电镜（SP cro - em）快速确定G蛋白偶联受体（GPCR）信号复合物的结构。生成了两种主要的G蛋白亚类：三聚体G(i)和G(s)，以及mini-G(s)，并进行了测试，以确保在其同源gpcr的背景下结合。表位排序显示每个G（α - β - γ）蛋白存在多个不同的表位。在三聚体G(i)和G(s)之间发现了几种G β γ特异性sABs，表明它们可以以“即插即用”的方式用于所有子类。还生成了代表另一类GPCR信号伙伴G蛋白受体激酶1 （GRK1）的单克隆抗体，并进行了进一步评估，支持该方法的普遍性。EM数据表明，亚类特异性sABs为多个GPCR信号复合物提供了有效的单基准和双基准。
"Universal'' synthetic antibody (sAB)-based fiducial marks have been generated by customized phage display selections to facilitate the rapid structure determination of G protein-coupled receptor (GPCR) signaling complexes by single-particle cryo-electron microscopy (SP cryo-EM). sABs were generated to the two major G protein subclasses: trimeric G(i) and G(s), as well as mini-G(s), and were tested to ensure binding in the context of their cognate GPCRs. Epitope binning revealed that multiple distinct epitopes exist for each G(alpha beta gamma) protein. Several G beta gamma-specific sABs, cross-reactive between trimeric G(i) and G(s), were identified suggesting they could be used across all subclasses in a "plug and play'' fashion. sABs were also generated to a representative of another class of GPCR signaling partner, G protein receptor kinase 1 (GRK1) and evaluated further, supporting the generalizability of the approach. EM data suggested that the subclass-specific sABs provide effective single and dual fiducials for multiple GPCR signaling complexes.