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编码的约800 G蛋白偶联受体（gpcr）的直接信号调节因子。阻滞蛋白- gpcr复合物是高度动态的，对晶体学和低温电镜来说是具有挑战性的对象。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，并且通常携带大量的人工修饰。该项目的总体目标是确定β -抑制蛋白在哺乳动物细胞的自然环境中直接与完全糖基化和最低限度修饰的gpcr结合的拓扑特征。我们应用基因编码的非规范氨基酸进行广泛的光交联和化学交联。我们绘制了捕集蛋白gpcr复合物内的接近点，并确定了捕集蛋白受体氨基酸的分子间对，这些氨基酸位于相互接近的位置。我们将这些信息应用于分子建模实验，并建立了gpcr -阻滞蛋白相互作用的详细模型。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（PTH1R）之间的稳定相互作用。我们已经建立了pth1r - β -ar -1复合物的详细模型，该模型基于300多个实验约束。该模型揭示了在所有已发表的gpcr -阻滞蛋白结构中缺失的柔性受体部分，如环和c端区域的许多细节，并揭示了β -阻滞蛋白1区域，特别是n结构域的远端区域（n边缘）的未知作用。此外，我们证明了该方法适用于瞬时gpcr -阻滞蛋白相互作用，这是经典结构确定方法无法实现的。现在，我们想要澄清观察到的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。我们还想描述我们观察到的pth1r阻滞复合物的其他排列，这些排列既不符合模型中所代表的主要状态，也不符合任何已发表的结构。其次，我们想要描述抑制蛋白与没有高度磷酸化c尾的gpcr结合的特征，因为3D数据完全缺失。第三，我们想要研究不同GPCR激酶（GRKs）产生的磷酸化模式对抑制蛋白结合构象特征的影响，并表征瞬时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-抑制蛋白相互作用的理解，特别是GPCR磷酸化与GPCR结合的抑制蛋白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.