Three‐Dimensional Structure of the Highly Conserved Seventh Transmembrane Domain of G‐Protein‐Coupled Receptors

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Abstract
The S/T-X1-X2-N-P-X3-X4-Y highly conserved sequence of the seventh transmembrane (TM VII) segment of G-protein-coupled receptors is not present in the photon receptor bacteriorhodopsin TM VII domain. Despite this noticeable discrepancy in sequence, the X-ray structure of bacteriorhodopsin is generally used as the key structure for modelling all G-protein-coupled receptors. Thus, a kinked trans -Pro helix is usually accepted for the TM VII three-dimensional structure of G-protein-coupled receptors, although Asn-Pro dipeptide mainly induces a type I/III β-turn conformation in both model peptides and proteins. NMR studies in various solvents and molecular calculations were undertaken in order to gain insight into the conformational behaviour of a 15-residue peptide from the tachykinin NK-1 TM VII domain incorporating this common sequence. The low solubility of this membrane-embedded peptide precludes methanol or micellar systems mimicking membrane environment; thus only dimethylsulfoxide (Me2SO) or chlorofonn/Me2SO mixture could be used. We also found that perfluoro-tert -butanol, which has not been previously used for NMR studies, constitutes an excellent alternative solvent for the analysis of hydrophobic peptides. The postulated kinked trans -Pro helix was only present as a minor conformer in Me2SO and an equilibrium between helical and extended structures existed. From NOE data a type I/III β-structure, centered around Pro9-Ile10, probably stabilized by an Asx turn, may be postulated. Addition of chloroform in Me2SO increased the percentage of folded structures but no preferential conformation could be proposed. In perfluoro-tert -butanol/CD3OD (9:1) the N- and C-terminal regions presented an α-helical structure, and these two domains were linked by a hinge around Asn-Pro with a γ-turn for the preceding residue Tyr7 and either a type I/III β-turn around Pro9–Ile10 or αR orientations for these residues, which are both stabilized by an Asx turn. As determined by energy calculations, these structures were equally as stable as the kinked trans -Pro helix and could constitute key structures for analysing the conformational changes and/or the dynamics of TM VII segment induced by the ligand when interacting with the receptor.