Transient ligand contacts of the intrinsically disordered N-terminus of neuropeptide Y2 receptor regulate arrestin-3 recruitment

Previous efforts in delineating molecular mechanisms of G protein-coupled receptor (GPCR) activation have focused on transmembrane regions and ligand-receptor contacts of the extracellular loops. The role of the highly flexible N-termini of rhodopsin-like GPCRs have not been well characterized to date. We hypothesize that transient contacts between the peptide ligand and the intrinsically disordered N-terminus (NT) of the neuropeptide Y (NPY) receptor Y₂ (Y₂R) will affect receptor signaling. We employ cross-linking mass spectrometry to capture ligand-receptor contacts including transient binding modes. A photo-reactive NPY analogue allows mapping the interaction between NPY and Y₂R NT resulting in a total number of 40 cross-links. The cross-links provide distance constraints for deriving structural models of the interaction. Molecular dynamics simulations highlight the structural flexibility and rapid interconversion of ligand-receptor contacts. Mutagenesis of Y₂R and functional characterization suggest that the cross-linking hotspots in the NT electrostatically control its conformational ensemble. The NT engages in transient contacts to the peptide and prolongs ligand residence time, which is required for efficient interaction of Y₂R with arrestin-3, but not G_i. We delineate structure-function relationships for the intrinsically disordered Y₂R NT and propose a functional role for transient binding modes involving the NT of a peptide-binding receptor.