panoramaProteomicsJuan Camilo Rojas EcheverriHomo SapiensEscherichia ColiSus Scrofahttps://panoramaweb.org/XL-MS_Y2R-NPY_photoLeu.urljuank1892@gmail.comResearch AssistantPanoramaPublicPrevious 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.Transient ligand contacts of the intrinsically disordered N-terminus of neuropeptide Y₂ receptor regulate arrestin-3 recruitment.Kaiser Anette A, Rojas Echeverri Juan C JC, Baischew Asat A, Pankonin Maik M, Leitner Karl D KD, Iacobucci Claudio C, Sala Davide D, Ihling Christian C, Müller Ronny R, Ferenc Rok R, Beck-Sickinger Annette G AG, Schmidt Peter P, Meiler Jens J, Hildebrand Peter W PW, Sinz Andrea AfalseMolecular details of ligand binding to the intrinsically disordered N terminus of the neuropeptide Y receptorNeuropeptide Y (NPY) receptors comprise a family of rhodopsin-like G-protein coupled receptors (GPCRs) that participate in controlling food intake, memory retention, and circadian rhythm, making them highly attractive drug targets. However, the multiligand nature of NPY receptors, such as the NPY receptor type 2 (Y2R), requires a detailed understanding of the receptor’s interactions with their natural ligands. Binding assays have so far provided insights into multiple Y2R-NPY conformers with distinct binding affinities that might also be controlled by intracellular Y2R-Gi protein-protein interactions. However, structural biology approaches, such as X-ray crystallography or cryo-electron microscopy, have so far not been able to capture these Y2R-NPY conformers, nor have they been able to resolve the conformational states of Y2R’s N-terminus. The N-terminus of Y2R has been classified as intrinsically disordered region. Cross-linking mass spectrometry (XL-MS) was employed to characterize the different conformational states and binding modes of Y2R upon binding of its ligand NPY. Cross-linked peptide digests were analyzed on a timsTOF Pro instrument using Parallel Signal Accumulation and Fragmentation operated in data dependent acquisition (DDA) and data independent acquisition (DIA) modes. The combined effects of precursor ion accumulation in the TIMS cell, fragment ion current accumulation through fragmentation events stacking, and the additional ion mobility dimension of peptide separation by DDA-PASEF provided better fragment ion spectral evidence of low-intensity cross-linked peptides. At the same time, spectra generated by co-isolation of high-abundant unmodified peptides were minimized. The comprehensive evaluation of each cross-linked peptide’s chromatographic and ion mobility properties in Skyline, in addition to fragment ion spectra, greatly improved the confidence of filtered peptide identifications. For the Y2R-NPY interaction, multiple cross-linking sites were identified between Y2R and NPY, involving the disordered N-terminal region of Y2R. The coexistence of different cross-linking sites between Y2R’s N-terminus and different amino acids in NPY point to multiple conformational states of Y2R’s N-terminus. Our results provide first insights into the interactions of NPY with Y2R at a molecular level.Tue Mar 24 00:00:00 GMT 2026PXD0518655629606982340973726