Three-dimensional structure of the highly conserved seventh transmembrane domain of G-protein-coupled receptors.
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 beta-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 chloroform/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 beta-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 alpha-helical structure, and these two domains were linked by a hinge around Asn-Pro with a gamma-turn for the preceding residue Tyr7 and either a type I/III beta-turn around Pro9-Ile10 or alpha R 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.
Laboratoire de Chimie Organique Biologique, CNRS URA 493, Université Pierre et Marie Curie, Paris, France.
J P
JP
Berlose
O
O
Convert
A
A
Brunissen
G
G
Chassaing
S
S
Lavielle
D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000737:chemistry; D017124:Conserved Sequence; D019204:GTP-Binding Proteins / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013816:Thermodynamics
D010455:Peptides; D011956:Receptors, Cell Surface; D001436:Bacteriorhodopsins; D019204:GTP-Binding Proteins