Structural Dynamics of the Ligand-Receptor Interaction of the Neuropeptide Y Receptor Type 2

Abstract

The molecular dynamics of the neuropeptide Y2 receptor was investigated by solid-state NMR. Quantitative static 15N NMR spectra and determination of 1H-13C order parameters through measurement of the 1H-13C dipolar couplings revealed axially symmetric motions of the whole molecule and molecular fluctuations of varying amplitude from all molecular segments. The molecular order parameters (S(backbone) = 0.59-0.67, S(CH2) = 0.41-0.51 and S(CH3) = 0.22) demonstrate that the Y2 receptor is highly mobile in the membrane. The receptor was found to be more rigid in monounsaturated POPC membranes than in saturated DMPC. This could be caused by an increased chain length of the monounsaturated lipids, which may result in a higher helical content of the receptor. Furthermore, cholesterol, phosphatidylethanolamine, or negatively charged phosphatidylserine did not have a significant influence on the molecular mobility of the Y2 receptor. We also developed a structural model of neuropeptide Y (NPY) bound to the receptor. Isotope-labeled NPY was used to determine the secondary structure of the ligand. Upon binding, the C-terminal α helix of NPY is unwound starting at T32 to make optimal contact of the C terminal residues within binding pocket. In addition, signals of several hydrophobic residues in the α-helical region of NPY were broadened upon receptor binding. These experimental data were used to derive a model of the Y2 receptor with the docked ligand, which was verified by double-cycle mutagenesis. The ligand is tethered to the second extracellular loop by hydrophobic contacts, with the N-terminal part of its helix facing the solvent. The C terminal pentapeptide of NPY inserts deeply into the transmembrane bundle, making optimal contacts to the receptor including an interaction of NPY's amidated C terminus with Q3.32 in a polar cluster within transmembrane helices 2 and 3.