Determining topology of beta-arrestin binding to non-visual G-protein coupled receptors in live cells via genetic incorporation of chemical tools

通过化学工具的遗传掺入确定活细胞中 β-arrestin 与非视觉 G 蛋白偶联受体结合的拓扑结构

• 批准号: 316443431
• 负责人: Professorin Dr. Irene Coin
• 金额: --
• 依托单位: Arbeitsgruppe Synthetische Proteinbiochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/316443431?language=en
• 关键词: Determining; topology; beta; arrestin; binding

Beta-arrestins are direct signalling regulators of the about 800 G protein-coupled receptors (GPCRs) encoded in the human genome. Arrestin-GPCR complexes are highly dynamic and represent challenging objects for crystallography and cryo-EM. The few structures that have been solved so far all include receptors that feature a highly phosphorylated C-tail and carry often heavy artificial modifications. General aim of this project is to identify topological features of beta-arrestin binding to fully-glycosylated and minimally modified GPCRs directly in the natural context of the live mammalian cell. We apply genetically encoded non-canonical amino acids for extensive photo- and chemical crosslinking. We map proximity points within arrestin-GPCR complexes and pin-point intermolecular pairs of arrestin-receptor amino acids that lie in reciprocal proximity. We apply this information to molecular modelling experiments and build detailed models for the GPCR-arrestin interaction. In the first funding period, we used this strategy to characterize the stable interaction between beta-arrestin-1 and the parathyroid hormone receptor (PTH1R). We have built a detailed model for the PTH1R-beta-arr-1 complex that is based on more than three hundred experimental constraints. The model reveals a number of details in flexible receptor parts like loops and C-terminal region that are missing in all published GPCR-arrestin structures and reveals a hereto unknown role of beta-arrestin-1 regions, especially the distal region of the N-domain (N-edge). Moreover, we demonstrate that the approach is applicable to transient GPCR-arrestin interactions, which are not accessible to classical structure determination methods. Now, we want to clarify whether the observed role of the N-edge is a conserved feature for different GPCRs and possibly also for beta-arrestin-2. We want also to characterize alternative arrangements of the PTH1R-arrestin complex that we have observed, and that are not compatible either with the predominant state represented in the model or with any of the published structures. In the second place, we want to characterize arrestin binding to GPCRs that do not have a highly phosphorylated C-tail, for which 3D data are completely missing. Third, we want to investigate the effect of phosphorylation patterns generated by distinct GPCR kinases (GRKs) to conformational features of arrestin binding, as well as characterize transient GPCR-arrestin complexes. We expect our data will contribute to complete the understanding of the GPCR-arrestin interaction and in particular the connection between GPCR phosphorylation and structural features of GPCR-bound arrestin.

β-抑制蛋白是人类基因组中编码的约800个G蛋白偶联受体（GPCR）的直接信号调节剂。Arrestin-GPCR复合物是高度动态的，代表了晶体学和cryo-EM的挑战性对象。到目前为止，已经解决的少数结构都包括具有高度磷酸化的C-尾的受体，并且通常进行大量的人工修饰。该项目的总体目标是在活哺乳动物细胞的自然环境中直接鉴定β-抑制蛋白与完全糖基化和最小修饰的GPCR结合的拓扑特征。我们应用遗传编码的非规范氨基酸进行广泛的光和化学交联。我们映射内的arrestin-GPCR复合物和针点arrestin-receptor氨基酸的相互接近的分子间对接近点。我们将这些信息应用于分子建模实验，并建立详细的GPCR-抑制蛋白相互作用的模型。在第一个资助期，我们使用这种策略来表征β-arrestin-1和甲状旁腺激素受体（PTH 1 R）之间的稳定相互作用。我们已经建立了一个详细的模型PTH 1 R-beta-β-1复合物，是基于300多个实验的限制。该模型揭示了在所有已发表的GPCR-抑制蛋白结构中缺失的柔性受体部分如环和C-末端区域中的许多细节，并揭示了迄今未知的β-抑制蛋白-1区域的作用，特别是N-结构域的远端区域（N-边缘）。此外，我们证明，该方法适用于瞬态GPCR抑制蛋白的相互作用，这是无法访问经典的结构测定方法。现在，我们想弄清楚观察到的N边的作用是否是不同GPCR的保守特征，也可能是β-arrestin-2的保守特征。我们还希望表征我们观察到的PTH 1 R-抑制蛋白复合物的替代排列，这些排列与模型中的主导状态或任何已发表的结构都不兼容。其次，我们希望表征抑制蛋白与不具有高度磷酸化的C-尾的GPCR的结合，其中3D数据完全缺失。第三，我们想研究不同的GPCR激酶（GRKs）产生的磷酸化模式对抑制蛋白结合的构象特征的影响，以及表征瞬时GPCR-抑制蛋白复合物。我们希望我们的数据将有助于完成对GPCR-arrestin相互作用的理解，特别是GPCR磷酸化和GPCR结合的arrestin结构特征之间的联系。