Genetically Encoded Chemical Probes in Cells Reveal the Binding Path of Urocortin-I to CRF Class B GPCR

Molecular determinants regulating the activation of class B G-protein-coupled receptors (GPCRs) by native peptide agonists are largely unknown. We have investigated here the interaction between the corticotropin releasing factor receptor type 1 (CRF1R) and its native 40-mer peptide ligand Urocortin-I directly in mammalian cells. By incorporating unnatural amino acid photochemical and new click-chemical probes into the intact receptor expressed in the native membrane of live cells, 44 intermolecular spatial constraints have been derived for the ligand-receptor interaction. The data were analyzed in the context of the recently resolved crystal structure of CRF1R transmembrane domain and existing extracellular domain structures, yielding a complete conformational model for the peptide-receptor complex. Structural features of the receptor-ligand complex yield molecular insights on the mechanism of receptor activation and the basis for discrimination between agonist and antagonist function. [Display omitted] •Ucn1 binding interface on CRF1R mapped with photochemical probes in cells•Position of Ucn1 in CRF1R determined with a click-chemical probe in cells•Conformational model for ligand-GPCR complex satisfies ∼50 spatial constraints•Insights on class B GPCR activation gained on receptor expressed in native cells A class B GPCR was systematically engineered to permit crosslinking to its native ligand in cells, and the resultant distance constraints allow modeling agonist binding and receptor activation.