Structural details of a Class B GPCR-arrestin complex revealed by genetically encoded crosslinkers in living cells

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. The conformation of GPCR-arrestin complexes at the cell membrane, despite available structures, remains uncertain. This work reveals structure and dynamics of the PTH1R-arrestin2 complex, including flexible regions, in live cells.

Automated summary

Understanding the molecular basis of arrestin-mediated regulation of GPCRs is crucial for deciphering signaling mechanisms. However, structural studies are challenging due to the dynamic nature of GPCR-arrestin complexes. In this study, the interaction between arrestin-2 and PTH1R was dissected using crosslinking amino acids in live cells. The integrative approach revealed the structure, dynamics, and flexible regions of the PTH1R-arrestin2 complex.