3-DIMENSIONAL STRUCTURE OF THE HIGHLY CONSERVED 7TH TRANSMEMBRANE DOMAIN OF G-PROTEIN-COUPLED RECEPTORS

The S/T-X(1)-X(2)-N-P-X(3)-X(4)-Y highly conserved sequence of the sev enth transmembrane (TM VII) segment of G-protein-coupled receptors is not present in the photon receptor bacteriorhodopsin TM VII domain. De spite 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 us ually accepted for the TM VII three-dimensional structure of G-protein -coupled receptors, although Asn-Pro dipeptide mainly induces a type I /III beta-turn conformation in both model peptides and proteins. NMR s tudies in various solvents and molecular calculations were undertaken in order to gain insight into the conformational behaviour of a 15-res idue 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 (Me(2)SO) or chloroform/Me(2)SO mixture could be used. We also found that perfluoro-tert-butanol, which has no t been previously used for NMR studies, constitutes an excellent alter native solvent for the analysis of hydrophobic peptides. The postulate d kinked trans-Pro helix was only present as a minor conformer in Me(2 )SO and an equilibrium between helical and extended structures existed . From NOE data a type I/III beta-structure, centered around Pro9-Ile1 0, probably stabilized by an Asx turn, may be postulated. Addition of chloroform in Me(2)SO increased the percentage of folded structures bu t no preferential conformation could be proposed. In perfluoro-tert-bu tanol/CD3OD (9:1) the N- and C-terminal regions presented an alpha-hel ical structure, and these two domains were linked by a hinge around As n-Pro with a gamma-turn for the preceding residue Tyr7 and either a ty pe I/III beta-turn around Pro9-Ile10 or alpha(R) orientations for thes e residues, which are both stabilized by an Asx turn. As determined by energy calculations, these structures were equally as stable as the k inked trans-Pro helix and could constitute key structures for analysin g the conformational changes and/or the dynamics of TM VII segment ind uced by the ligand when interacting with the receptor.