Neuropeptide 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.

Photo-reactive amino acids, i.e. photo-leucine, containing diazirine groups were incorporated at defined positions into NPY to undergo cross-linking reactions upon activation with UV-A irradiation. Cross-linking experiments were conducted between NPY and the Y2 receptor, incorporated in phospholipid bicelles, as well as the N-terminal 35 amino acids of Y2R (NT Y2R). Cross-linking reaction mixtures were irradiated with UV-A light and enzymatically digested with AspN and trypsin. Afterwards, cross-linked peptides were analyzed by LC-MS/MS using a tims-TOF Pro mass spectrometer (Bruker Daltonik) operating in data-dependent acquisition (DDA) and data independent acquisition (DIA) modes with parallel accumulation-serial fragmentation (PASEF). DDA-PASEF data were pre-processed with DataAnalysis (Bruker Daltonik) to generate peak lists of fragment ion spectra with metadata annotation of the ion mobility of the precursor ions. Fragment ion mass spectra were interpreted with MeroX (v 2.0.1.7) to obtain initial cross-link spectral matches (XSMs) as well as with SearchGUI and Peptideshaker for the identification of unmodified peptides. Validated XSMs served as basis for a list of target peptides that, together with ion mobility data, allowed to create extracted ion chromatograms with Skyline-daily (v 23.1.1.459). Raw DDA PASEF and DIA-PASEF files were inspected with Skyline, comparing the chromatographic and ion mobility behavior of each cross-link with negative control samples, i.e. Y2R samples without NPY.

A cysteine-deficient variant of human Y2R was recombinantly expressed in E. coli as inclusion bodies using a fed-batch fermentation process, solubilized, and purified in the presence of sodium dodecyl sulfate (SDS), and functionally reconstituted in bicelles according to an existing protocol. The N-terminal 35 amino acids of human Y2R were synthesized using microwave-assisted automated synthesis. Porcine NPY variants were generated by peptide synthesis using a combined automated/manual synthesis on solid support using an Fmoc/tBu strategy. A photo reactive NPY variant was generated with a triple substitution. Specifically, leucine residues at positions 17, 24, and 30 were replaced by the diazirine-containing amino acid photo-leucine.