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

β-抑制素是人类基因组中编码的约800G蛋白偶联受体(GPCRs)的直接信号调节因子。Arrestin-GPCR复合体是高度动态的，是结晶学和低温EM具有挑战性的对象。到目前为止已经解决的为数不多的结构都包括以高度磷酸化的C-尾巴为特征的受体，并且通常带有大量的人工修饰。该项目的总体目标是在哺乳动物活细胞的自然环境中直接鉴定β-arrestin与完全糖基化和最小修饰的GPCRs结合的拓扑特征。我们应用基因编码的非标准氨基酸进行广泛的光和化学交联。我们在Arrestin-GPCR复合体和Arrestin-受体氨基酸的针点分子间对中绘制了相互接近的接近点。我们将这些信息应用于分子模拟实验，并为GPCR-arrestin相互作用建立了详细的模型。在第一个资助期，我们使用这一策略来表征β-arrestin-1和甲状旁腺激素受体(PTH1R)之间的稳定相互作用。我们已经建立了PTH1R-β-ARR-1复合体的详细模型，该模型基于300多个实验约束。该模型揭示了柔性受体部分的一些细节，如环和C-末端区域，这些细节在所有已发表的GPCR-arrestin结构中都缺失，并揭示了β-arrestin-1区域，特别是N-结构域的远端区域(N-边)迄今未知的作用。此外，我们还证明了该方法适用于经典结构确定方法所不能获得的GPCR-arrestin的瞬时相互作用。现在，我们想要澄清观察到的N-边的作用是否对不同的GPCR以及可能对β-arrestin-2来说是一个保守的特征。我们还想表征我们观察到的PTH1R-arrestin络合物的替代排列，这些排列与模型中表示的主要状态或任何已发表的结构都不相容。其次，我们想要表征arrestin与GPCRs的结合，这些GPCRs没有高度磷酸化的C-尾巴，其3D数据完全缺失。第三，我们想要研究不同的GPCRKs(GRKs)产生的磷酸化模式对arrestin结合的构象特征的影响，以及瞬时GPCRarrestin复合体的特征。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磷酸化与GPCRarrestin结构特征之间的联系。

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.