Unwinding of the C‐Terminal Residues of Neuropeptide Y is critical for Y₂ Receptor Binding and Activation

Abstract

Despite recent breakthroughs in G protein-coupled receptor (GPCR) structure characterization, there is only sparse data on how GPCRs recognize larger peptide ligands. Here, we integrate nuclear magnetic resonance (NMR) spectroscopy, molecular modeling, and double-cycle mutagenesis studies to obtain a structural model of the peptide hormone neuropeptide Y (NPY), bound to its human G protein-coupled Y2 receptor (Y2R). Solid-state NMR measurements of specifically isotope-labeled NPY in complex with in vitro folded Y2R reconstituted into phospholipid bicelles provide the bioactive structure of the peptide. Guided by solution NMR experiments, we find that the ligand is tethered to the second extracellular loop by hydrophobic contacts. The C-terminal α-helix of NPY, which is formed in membrane environment in the absence of the receptor, is unwound starting at T32 to provide optimal contacts in a deep binding pocket within the transmembrane bundle of the Y2R.

Structural basis of ligand binding modes at the neuropeptide Y Y1 receptor

“…In contrast, the NPY-bound model shows that side chain of Q120 3.32 points almost in the opposite direction and engages in a hydrogen bond with the hydroxy group of Y36-NH 2 (Extended Data Fig. 1d), similar as the interaction between the Y 2 R residue Q 3.32 and NPY suggested by Xu et al 28 In Y 2 R, it was also reported that Q 3.32 may interact with the C-terminal amide of NPY 16 . IP accumulation studies show that the Y 1 R mutation Q120 3.32 H leads to a 26-fold decrease in potency of NPY, and NPY-tyramide lacking the C-terminal amide displays a 45-fold loss of activity.…”

“…NPY-tyramide was synthesized as previously described 44 . Isotopically labelled NPY variants were prepared as described 16 , and 13 C/ 15 N labelled amino acids were coupled manually with 2 eq. HOBT/DIC in DMF over night.…”

“…Fourteen differently isotopically labelled (U- 13 C/ 15 N) porcine NPY peptides were prepared by standard Fmoc solid phase synthesis as described previously 16 . On the basis of the structure of micelle-bound NPY, the positions of the NMR labels were chosen to avoid signal overlap in 13 C- 13 C single quantum/double quantum correlation experiments and to allow straightforward signal assignment.…”

“…Together, these data suggest that the N terminus and ECL2 of Y 1 R play important roles in recognition of NPY’s N terminus and receptor activation. This contrasts with NPY binding at Y 2 R, in which ECL2 may interact with the central α-helix of NPY and the peptide N terminus is flexible and unanchored by the receptor 16 . Whereas this study provides insights into the interactions between Y 1 R and NPY, further structural details, such as the structures of Y receptors bound to NPY, are required to fully understand the endogenous agonist binding modes of the NPY receptor family.…”

“…In Y 1 R and Y 2 R, Q120 3.32 is suggested to be the interaction partner for the C terminus of NPY and crucial for receptor activation 16 . In the Y 1 R–UR-MK299 structure, this residue forms a hydrophobic contact with the phenyl ring of the hydroxyphenyl group in UR-MK299, potentially blocking the binding of Y 1 R to NPY.…”