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 (PTH1R) has been st ructurally characterized by NMR and molecular dynamics simulations. Th e fragment PTH1R[168-198] includes residues 173-189, shown by photoaff inity cross-linking to be a contact domain with position 13 of parathy roid hormone (PTH). The structure of PTH1R[168-198], determined in a m icellar solution of dodecylphosphocholine to mimic the membrane enviro nment, consists of three a-helices, separated by a well-defined turn a nd a flexible region. The topological orientation of PTH1R[168-198] wa s 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 d omain, is very hydrophobic and embedded in the lipid core. This helix is preceded by a well-defined turn, forming an approximate 90 degrees bend, placing the other helices (residues 169-176 and 180-189), both o f which are amphipathic, on the surface of the micelle. In this orient ation, many hydrophilic residues of the receptor, including Glu(177), Arg(179), Arg(181), Glu(182), Asp(185), and Arg(186) are projecting to ward the solvent available to form complementary Coulombic interaction s with the polar residues of the principal binding domain of the ligan d (e.g., Arg(25), Lys(26), Lys(27), Asp(30), and His(32)). Given that the binding domain of PTH adopts an amphipathic alpha-helix which lies on the membrane, we visualize ligand binding as a two stage process i nvolving a nonspecific hydrophobic interaction of amphipathic helices with the membrane, followed by two-dimensional diffusion leading to hi ghly specific, ligand-receptor interaction.