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

Abstract

The S/T-XI-X,-N-P-X,-X,-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 truns-Pro helix is usually accepted for the TM VII three-dimensional structure of G-proteincoupled receptors, although Asn-Pro dipeptide mainly induces a type UIII /3-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 (Me,SO) or chlorofonn/Me,SO 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 truns-Pro helix was only present as a minor conformer in Me,SO and an equilibrium between helical and extended structures existed. From NOE data a type I/III ,&structure, centered around Pro9-Ile10, probably stabilized by an Asx turn, may be postulated. Addition of chloroform in Me,SO increased the percentage of folded structures but no preferential conformation could be proposed. In perfluoro-tert-butanoI/CD,OD (9 : 1) the N-and C-terminal regions presented an a-helical structure, and these two domains were linked by a hinge around Asn-Pro with a y-turn for the preceding residue Tyr7 and either a type I/III p-turn around Pro9-IlelO or cxR 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 truns-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.The apparent similarity in the hydropathicity profiles of G-protein-coupled receptors [ 1 -41 with that of bacteriorhodopsin [5] has led to the use of this integral membrane protein as a starting point for predicting their three-dimensional structures [6-121, even though bacteriorhodopsin is not coupled to G-proteins. The currently accepted proposal for the topography of G-protein-coupled receptors includes seven transmembrane-spanning domains (TM I-VII) joined together by extracellular and cytoplasmic loops, with the Nterminal side in the extracellular cleft and the C-terminal residues in the cytoplasm. This model, which was first deduced Correspondence to S .

Molecular Dynamics Simulation of Membranes and a Transmembrane Helix

“…However, this latter HB is satisfied by a water molecule that has penetrated the membrane deeply enough to provide an hydrogen bond donor to this backbone carbonyl oxygen about 50% of the time (Figs. 13 and 14).It is significant that the main features of the TMS7 intramolecular hydrogen bonding pattern predicted by the MD simulations are in good agreement with the HBs deduced from the NMR data obtained on the tachykinin NK-1 helix 7 in a perfluoro-tert-butanol/CD 3 OD solvent [17]. In particular, the NH backbone group of residue ASN 8 in tachykinin NK-1, which corresponds to ASN 16 in TMS7, is predicted to form hydrogen bonds of type [i − (i + 2)] and [i − (i + 3)] with MET 6 and THR 5 backbone carbonyl oxygens.…”

“…In addition, it is found experimentally that the side chain of residue ASN 8 forms an HB with the proton of the NH group in ILE 10 as was found here between the residues ASN 16 and LEU 18 of the TMS7 peptide. Finally, it is noted that for both of these similar transmembrane segments, the backbone carbonyl oxygen of the proline tends to form an HB with the side chain of the fourth succeeding residue, which is respectively the SER 13 and THR 21 in the NK-1 and TMS7 peptides [17]. The average lengths of some of the crucial hydrogen bonds that stabilize the NP motif of TMS7 are reported in Table V [72], has a strong tendency to interact with the two preceding carbonyl oxygens of residues 12 and 13 to form more common [i − (i + 3)] and [i − (i + 4)] hydrogen bonds.…”

“…Among the methods used to study lipid bilayer structure, infra-red (IR) spectroscopy provides information about the trans to gauche ratio in the hydrocarbon chains [7,8], while x-ray and neutron diffraction methods provide information about the spatial distribution of the different groups of atoms relative to the membrane plane normal [9][10][11]. Nuclear magnetic resonance (NMR) has been used to characterize the internal conformations and dynamic behavior of the head groups and the acyl chains of lipid molecules [12][13][14][15], as well as the secondary structures and orientations of peptides embedded in bilayers [16][17][18][19]. The orientations of the acyl chains were probed in circular dichroism (CD) experiments [20,21] or IR spectroscopy [22,23].…”

Conformational and Associative Behaviours of the Third Helix of Antennapedia Homeodomain in Membrane‐Mimetic Environments

“…However due to the high molecular mass of the micellar aggregate, incomplete NH-to-sidechain correlations were observed for large side-chain amino acids such as Arg and Lys. Reverse correlations from either the 6 or e protons, compared with the connectivities observed in 0.2 -.......................................................................................................................................................................... (Bundi and Wuthrich, 1079) and in perfluoro-terr-butanol (Berlose et al, 1994).…”

“…5. The values for random coil structures have been determined for amino acids in model tetrapeptides trifluoroacetyl-GGXA-OCH, in aqueous solution (Bundi and Wiithrich, 1979) or Ac-GGXA-NH, (Berlose et al, 1994) in perfluro-tert-butanol solution. The CHa protons of residues in a-helical conformation or in a psheet conformation are shifted upfield or downfield, respectively (Wishart et al, 1991).…”