Binding domain of human parathyroid hormone receptor: From conformation to function

A 31 amino acid fragment of the extracellular N-terminus of the human G- protein coupled receptor for parathyroid hormone (PTHIR) has been structurally characterized by NMR and molecular dynamics simulations. The fragment PTHIR[168-198] includes residues 173-189, shown by photoaffinity cross-linking to be a contact domain with position 13 of parathyroid hormone (PTH). The structure of PTHIR[168-198], determined in a micellar solution of dodecylphosphocholine to mimic the membrane environment, consists of three α-helices, separated by a well-defined turn and a flexible region. The topological orientation of PTHIR[168-198] was determined from nitroxide- radical induced relaxation of NMR signals utilizing 5- and 16-doxylstearic acid. The C-terminal helix (residues 190- 196), consisting of seven amino acids of the first transmembrane domain, is very hydrophobic and embedded in the lipid core. This helix is preceded by a well-defined turn, forming an approximate 90°bend, placing the other helices (residues 169-176 and 180- 189), both of which are amphipathic, on the surface of the micelle. In this orientation, many hydrophilic residues of the receptor, including Glu¹⁷⁷, Arg¹⁷⁹, Arg¹⁸¹, Glu¹⁸², Asp¹⁸⁵, and Arg¹⁸⁶, are projecting toward the solvent available to form complementary Coulombic interactions with the polar residues of the principal binding domain of the ligand (e.g., Arg²⁵, Lys²⁶, Lys²⁷, Asp³⁰, and His³²). Given that the binding domain of PTH adopts an amphipathic α-helix which lies on the membrane, we visualize ligand binding as a two stage process involving a nonspecific hydrophobic interaction of amphipathic helices with the membrane, followed by two-dimensional diffusion leading to highly specific, ligand-receptor interaction.