Understanding the molecular basis of arrestin-mediated regulation of GPCRs is critical for deciphering signaling mechanisms and designing functional selectivity. However, structural studies of GPCR-arrestin complexes are hampered by their highly dynamic nature. Here, we dissect the interaction of arrestin-2 (arr2) with the secretin-like parathyroid hormone 1 receptor PTH1R using genetically encoded crosslinking amino acids in live cells. We identify 136 intermolecular proximity points that guide the construction of energy-optimized molecular models for the PTH1R-arr2 complex. Our data reveal flexible receptor elements missing in existing structures, including intracellular loop 3 and the proximal C-tail, and suggest a functional role of a hitherto overlooked positively charged region at the arrestin N-edge. Unbiased MD simulations highlight the stability and dynamic nature of the complex. Our integrative approach yields structural insights into protein-protein complexes in a biologically relevant live-cell environment and provides information inaccessible to classical structural methods, while also revealing the dynamics of the system.

了解抑制蛋白介导的 GPCR 调节的分子基础对于破译信号机制和设计功能选择性至关重要。然而，GPCR-arrestin 复合物的结构研究因其高度动态的性质而受到阻碍。在这里，我们使用基因编码的交联氨基酸在活细胞中剖析 arrestin-2 (arr2) 与分泌素样甲状旁腺激素 1 受体 PTH1R 的相互作用。我们确定了 136 个分子间邻近点，这些点指导 PTH1R-arr2 复合物的能量优化分子模型的构建。我们的数据揭示了现有结构中缺失的柔性受体元件，包括细胞内环 3 和近端 C 尾，并表明抑制蛋白 N 边缘迄今为止被忽视的带正电区域的功能作用。无偏见的 MD 模拟突出了复合体的稳定性和动态特性。我们的综合方法对生物学相关的活细胞环境中的蛋白质-蛋白质复合物产生了结构洞察力，并提供了经典结构方法无法获得的信息，同时还揭示了系统的动力学。