The pyrrolysyl-tRNA synthetase/tRNA^Pyl pair is the most versatile and widespread system for the incorporation of non-canonical amino acids (ncAAs) into proteins in mammalian cells. However, low yields of ncAA incorporation severely limit its applicability to relevant biological targets. Here, we generate two tRNA^Pyl variants that significantly boost the performance of the pyrrolysine system. Compared to the original tRNA^Pyl, the engineered tRNAs feature a canonical hinge between D- and T-loop, show higher intracellular concentrations and bear partially distinct post-transcriptional modifications. Using the new tRNAs, we demonstrate efficient ncAA incorporation into a G-protein coupled receptor (GPCR) and simultaneous ncAA incorporation at two GPCR sites. Moreover, by incorporating last-generation ncAAs for bioorthogonal chemistry, we achieve GPCR labeling with small organic fluorophores on the live cell and visualize stimulus-induced GPCR internalization. Such a robust system for incorporation of single or multiple ncAAs will facilitate the application of a wide pool of chemical tools for structural and functional studies of challenging biological targets in live mammalian 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 activation mechanism of class B G-protein-coupled receptors (GPCRs) remains largely unknown. To characterize conformational changes induced by peptide hormones, we investigated interactions of the class B corticotropin-releasing factor receptor type 1 (CRF1R) with two peptide agonists and three peptide antagonists obtained by N-truncation of the agonists. Surface mapping with genetically encoded photo-crosslinkers and pair-wise crosslinking revealed distinct footprints of agonists and antagonists on the transmembrane domain (TMD) of CRF1R and identified numerous ligand-receptor contact sites, directly from the intact receptor in live human cells. The data enabled generating atomistic models of CRF- and CRF(12-41)-bound CRF1R, further explored by molecular dynamics simulations. We show that bound agonist and antagonist adopt different folds and stabilize distinct TMD conformations, which involves bending of helices VI and VII around flexible glycine hinges. Conservation of these glycine hinges among all class B GPCRs suggests their general role in activation of these receptors.