Produção Nacional
Revisado por pares
Structure of Two Fragments of the Third Cytoplasmic Loop of the Rat Angiotensin II AT1A Receptor
1999; Elsevier BV; Volume: 274; Issue: 1 Linguagem: Inglês
10.1074/jbc.274.1.227
ISSN1083-351X
AutoresLorella Franzoni, Giuseppe Nicastro, Thelma A. Pertinhez, Eliandre de Oliveira, Clóvis R. Nakaie, Antonio C.M. Paiva, Shirley Schreier, Alberto Spisni,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoThe structural bases that render the third intracellular loop (i3) of the rat angiotensin II AT1Areceptor one of the cytoplasmic domains responsible for G-protein coupling are still unknown. The three-dimensional structures of two overlapping peptides mapping the entire i3 loop and shown to differently interact with purified G-proteins have been obtained by simulated annealing calculations, using NMR-derived constraints collected in 70% water/30% trifluoroethanol solution. While the NH2-terminal half, Ni3, residues 213–231, adopts a stable amphipathic α-helix, extending over almost the entire peptide, a more flexible conformation is found for the COOH-terminal half, Ci3, residues 227–242. For this peptide, a cis-transisomerization around the Lys6—Pro7 peptide bond generates two exchanging isomers adopting similar conformations, with an α-helix spanning from Asn9 to Ile15and a poorly defined NH2 terminus. A quite distinct structural organization is found for the sequence EIQKN, common to Ni3 and Ci3. The data do suggest that the extension and orientation of the amphipathic α-helix, present in the proximal part of i3, may be modulated by the distal part of the loop itself through the Pro233 residue. A molecular model where this possibility is considered as a mechanism for G-protein selection and coupling is presented. The structural bases that render the third intracellular loop (i3) of the rat angiotensin II AT1Areceptor one of the cytoplasmic domains responsible for G-protein coupling are still unknown. The three-dimensional structures of two overlapping peptides mapping the entire i3 loop and shown to differently interact with purified G-proteins have been obtained by simulated annealing calculations, using NMR-derived constraints collected in 70% water/30% trifluoroethanol solution. While the NH2-terminal half, Ni3, residues 213–231, adopts a stable amphipathic α-helix, extending over almost the entire peptide, a more flexible conformation is found for the COOH-terminal half, Ci3, residues 227–242. For this peptide, a cis-transisomerization around the Lys6—Pro7 peptide bond generates two exchanging isomers adopting similar conformations, with an α-helix spanning from Asn9 to Ile15and a poorly defined NH2 terminus. A quite distinct structural organization is found for the sequence EIQKN, common to Ni3 and Ci3. The data do suggest that the extension and orientation of the amphipathic α-helix, present in the proximal part of i3, may be modulated by the distal part of the loop itself through the Pro233 residue. A molecular model where this possibility is considered as a mechanism for G-protein selection and coupling is presented. G-protein coupled receptors type 1A angiotensin II receptor third intracellular loop of AT1A NH2-terminal fragment 213–231 and COOH-terminal fragment 227–242 of the third intracellular loop 2,2,2-trifluoroethanol nuclear Overhauser effect two-dimensional NOE spectroscopy transmembrane helix. The comprehension of the molecular details of G-protein coupled receptors (GPCRs)1 activation as well as of G-protein selection and coupling is still speculative. Similarly, several features of their three-dimensional structure still need to be defined. In this respect, while a large effort is being generated to define the orientation and three-dimensional organization of the transmembrane helices of GPCRs (1Hargrave P.A. Curr. Opin. Struct. Biol. 1991; 1: 575-581Crossref Scopus (33) Google Scholar, 2Baldwin J.M. EMBO J. 1993; 12: 1693-1703Crossref PubMed Scopus (886) Google Scholar, 3Baldwin J.M. Curr. Opin. Cell Biol. 1996; 6: 180-190Crossref Scopus (341) Google Scholar, 4Donnelly D. 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Soc. 1996; 118: 8998-9004Crossref Scopus (40) Google Scholar) have prompted us to undertake a study on some functionally relevant cytoplasmic domains of the angiotensin II AT1A receptor, mainly seeking to describe the structural and dynamic properties of the receptor surface that regulate its interaction with the various G-proteins. In a previous work, we focused our attention on the conformational flexibility of a fragment of the AT1A COOH-terminal tail (17Franzoni L. Nicastro G. Pertinhez T.A. Tatò M. Nakaie C.R. Paiva A.C.M. Schreier S. Spisni A. J. Biol. Chem. 1997; 272: 9734-9741Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar); here we show the existence and propose a model describing the dynamic features of the structural determinants that characterize the receptor third intracellular loop (i3) (Fig.1). As for various GPCRs such as the β-adrenergic, muscarinic, dopamine, and rhodopsin receptors (18Hausdorff W.P. Hnatowich M. O'Dowd B.F. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1990; 265: 1388-1393Abstract Full Text PDF PubMed Google Scholar, 19Guan X.-M. Amend A. Strader C.D. Mol. Pharmacol. 1995; 48: 492-498PubMed Google Scholar, 20Eason M.G. Liggett S.B. J. Biol. Chem. 1996; 271: 12826-12832Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 21Tseng M.-J. Coon S. Stuenkel E. Struk V. Logsdon C.D. J. Biol. Chem. 1995; 270: 17884-17891Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 22Guiramand J. Montmayeur J.-P. Ceraline J. Bhatia M. Borrelli E. J. Biol. Chem. 1995; 270: 7354-7358Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 23Thurmod L.R. Creuzenet C. Reeves J.P. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 94: 1715-1720Crossref Scopus (43) Google Scholar), studies involving receptor chimeras and site-directed mutagenesis (24Wang C. Jayadev S. Escobedo J.A. J. Biol. Chem. 1995; 270: 16677-16682Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 25Conchon S. Barrault M.-B. Miserey S. Corvol P. Clauser E. J. Biol. Chem. 1997; 272: 25566-25572Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 26Thompson J.B. Wade S.M. Harrison J.K. Salafranca M.N. Neubig R.R. J. Pharmacol. Exp. Ther. 1998; 285: 216-222PubMed Google Scholar, 27Zhu B.M. Neubig R.R. Wade S.M. Posner P. Gelband C.H. Summer C. Am. J. Physiol. 1997; 273: 1040-1048Crossref PubMed Google Scholar, 28Kai H. Alexander R.W. Ushio-Fukai M. Lyons P.R. Akers M. Griendling K.K. Biochem. J. 1998; 332: 781-787Crossref PubMed Scopus (27) Google Scholar) indicate that i3 is one of the AT1A functional domains involved in G-protein interaction. In addition, comparison of the i3 of several GPCRs evidences a noticeable heterogeneity in amino acid sequence and size (29Wess J. Brann M.R. Bronner T.I. FEBS Lett. 1989; 258: 133-136Crossref PubMed Scopus (80) Google Scholar, 30Cotecchia S. Ostrowski J. Kjelsberg A.M. Caron G.M. Lefkowitz J.R. J. Biol. Chem. 1992; 267: 1633-1639Abstract Full Text PDF PubMed Google Scholar, 31Sasaki K. Yamano Y. Bardhan S. Iwai N. Murray J.J. Hasegawa M. Matsuda Y. Inagami T. Nature. 1991; 351: 230-233Crossref PubMed Scopus (778) Google Scholar, 32Murphy T.J. Alexander R.W. Griendling K.K. Runge M.S. Bernstein K.E. Nature. 1991; 351: 233-236Crossref PubMed Scopus (1171) Google Scholar), suggesting that the secondary structure, rather than the primary sequence and/or the specific length of that domain, plays a key role in G-protein coupling. Recent investigations revealed that a synthetic peptide representing the proximal part of i3 (residues 216–230) is able to activate purified Gi and G0 proteins, while the peptide comprising the distal part of that loop (residues 229–242) has no effect (33Shirai H. Takahashi K. Katada T. Inagami T. Hypertension. 1995; 25: 726-730Crossref PubMed Google Scholar). Similar results have been obtained in a study focused on the activation of Gq, where it has been shown that the peptide encompassing residues 216–231 is active, while the one representing the i3 segment 230–241 does not exhibit any activity (34Sano T. Ohyama K. Yamano Y. Nakagomi Y. Nakazawa S. Kikyo M. Shirai H. Blank J.S. Exton J.H. Inagami T. J. Biol. Chem. 1997; 272: 23631-23636Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Interestingly, the proximal half of i3 has been predicted to have a very high probability to adopt an amphipathic α-helical structure, whereas the distal half is predicted to form a short helix only at its COOH-terminal end (35Rost B. Sander C. Proteins. 1994; 19: 55-72Crossref PubMed Scopus (1342) Google Scholar, 36Rost B. Sander C. Schneider R. Compu. Appl. Biosci. 1994; 10: 53-60PubMed Google Scholar). On the basis of these evidences, we have studied the solution conformation of two synthetic fragments mapping the entire i3 loop, the NH2-terminal 19-mer TSYTLIWKALKKAYEIQKN-NH 2, Ni3 (residues 213–231), and the C COOH-terminal 16-merEIQKNKPRNDDIFRII-NH 2, Ci3 (residues 227–242). The underlined residues indicate the overlapping region between the two peptides. The structures of the two peptides in 70% H2O/30% TFE were obtained by means of restrained molecular dynamics calculations using, as restraints, the NMR-derived proton distances and φ dihedral angles. The results show that Ni3 is characterized by a well defined amphipathic α-helix extending over almost the entire peptide sequence. For Ci3, the data indicate the existence of cis-transisomerization about the Lys6—Pro7 peptide bond giving rise to two slowly exchanging conformational states. The resulting isomers adopt very similar secondary structures characterized by a poorly defined NH2 terminus and by a flexible amphipathic α-helix in the COOH-terminal stretch (Asn9-Ile15). Interestingly, the sequence EIQKN common to Ni3 and Ci3, and corresponding to the central part of i3, adopts a quite distinct structural organization in the two peptides suggesting, for Pro233, a functional role as structure breaker or modulator (37Chou P.Y. Fasman G.D. Adv. Enzymol. 1978; 47: 45-148PubMed Google Scholar, 38Richardson J.S. Richardson D.C. Science. 1988; 240: 1648-1652Crossref PubMed Scopus (1299) Google Scholar). The results of this work, together with our previous study (17Franzoni L. Nicastro G. Pertinhez T.A. Tatò M. Nakaie C.R. Paiva A.C.M. Schreier S. Spisni A. J. Biol. Chem. 1997; 272: 9734-9741Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), provide the basis for disclosing some conformational features of the native receptor cytosolic face. In particular, the data support the hypothesis that the capability of specific domains of the receptor to form amphipathic α-helices is essential for receptor activation and G-protein selection and coupling. The two peptides corresponding to residues 213–231 (Ni3) and 227–242 (Ci3) of the i3 loop of the rat angiotensin II AT1A receptor (Fig. 1) have been synthesized by the solid phase method usingt-butoxycarbonyl chemistry. Optimized coupling conditions were introduced according to the resin solvation theory (39Cilli E.M. Oliveira E. Marchetto R. Nakaie C.R. J. Org. Chem. 1996; 61: 8992-9000Crossref PubMed Scopus (75) Google Scholar). The peptides were purified by high pressure liquid chromatography, and their purity and molecular weight were confirmed by mass spectrometry. Fig. 5 shows the sequences with both the numbering for the residues of the peptides and the one from the primary sequence of rat AT1A receptor (32Murphy T.J. Alexander R.W. Griendling K.K. Runge M.S. Bernstein K.E. Nature. 1991; 351: 233-236Crossref PubMed Scopus (1171) Google Scholar). Far UV CD spectra were recorded on a Jasco J-715 spectropolarimeter using a Peltier system PTC-348 WVI for cell temperature control. Ellipticity is reported as the mean residue molar ellipticity, [θ] (deg cm2dmol−1). The instrument has been calibrated with recrystallized d-10-camphorsulfonic acid. The H2O/TFE cross-titration experiments were carried out mixing the appropriate aliquots of two 0.15 mm stock solutions, one in water at pH ≈ 4 and the other in TFE. A 1 mm cell was used. NMR samples were prepared in 70% H2O/30% TFE-d 3 (v/v), pH ≈ 4, to yield a peptide concentration of about 2 mm for both peptides. All two-dimensional 1H-NMR experiments were recorded and processed by the procedures elsewhere described (17Franzoni L. Nicastro G. Pertinhez T.A. Tatò M. Nakaie C.R. Paiva A.C.M. Schreier S. Spisni A. J. Biol. Chem. 1997; 272: 9734-9741Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). All calculations were carried out on a Silicon Graphics ONYX computer as described previously (17Franzoni L. Nicastro G. Pertinhez T.A. Tatò M. Nakaie C.R. Paiva A.C.M. Schreier S. Spisni A. J. Biol. Chem. 1997; 272: 9734-9741Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The structures of the two peptides were computed using the final set of NOE-derived distance constraints listed in Table III together with the φ dihedral angles. The quality of the final structures was verified on the basis of the minimum number of NOE distance and φ dihedral angle violations as well as of the minimum root mean square deviation values of the backbone atoms in the region of interest. For NOE distance and φ dihedral angle violations, an upper limit of 0.3 Å and 5 degrees has been used, respectively.Table II1H-NMR chemical shifts and 3JHN-α coupling constants for Ni3, (2.0 mm) in 70% H2O/30% TFE-d3 (v/v), pH ≈ 4, at 25 °CResidue3 J HN-αNHαHβHγHOthersHzppmThr13.884.081.24Ser28.664.603.95, 3.88Tyr36.28.304.422.97, 2.86δH 7.06; εH 6.82Thr45.67.784.054.211.29Leu56.27.674.221.80, 1.681.74δH 1.01, 0.94Ile66.57.503.861.94γH2 1.60, 1.22γH3 0.89; δH 0.86Trp75.68.064.433.36, 3.341NH 9.81; 2H 7.17; 4H 7.59; 5H 7.10; 6H 7.20; 7H 7.44Lys85.97.963.902.00, 1.931.44δH 1.73; εH 3.01Ala95.97.994.151.57Leu106.08.504.121.891.52δH0.86Lys115.78.154.001.82, 1.741.35δH 1.63; εH 2.95Lys126.27.854.162.04, 1.961.56δH 1.78; εH 2.99Ala135.68.194.101.54Tyr145.98.404.233.21δH7.15; εH 6.83Glu155.98.173.952.32, 2.212.64, 2.50Ile166.58.263.852.01γH2 1.60, 1.23γH3 0.95; δH 0.88Gln176.38.064.112.162.54, 2.38δNH2 7.30, 7.07Lys185.98.174.101.72, 1.691.33δH 1.62; εH 2.98Asn197.67.904.692.90, 2.81γNH2 7.61, 6.86term-NH2 7.16, 6.61 Open table in a new tab Table I1H-NMR chemical shifts and 3JHN-α coupling constants for the trans- and the cis-Pro-containing isomers of Ci3, (2.6 mm) in 70% H2O/30% TFE-d3 (v/v), pH ≈ 4 at 25 °CResidue3 J HN-αNHαHβHγHOthersHzppmGlu14.152.172.45Ile27.28.684.241.89γH2 1.55, 1.25γH3 0.98; δH 0.95Gln36.48.514.412.14, 2.042.43δNH2 7.56, 6.82Lys47.28.384.341.88, 1.781.50, 1.46δH 1.75; εH 3.04Asn56.48.364.602.80γNH27.58, 6.88Lys67.28.134.651.88, 1.781.51δH 1.76; εH 3.067.3aNumbers in italics, trans andcis-Pro-containing isomers.8.174.641.88, 1.791.51δH 1.76; εH 3.06Pro74.472.34, 1.962.07, 2.04δH 3.85, 3.684.462.35, 1.962.07, 2.04δH 3.85, 3.69Arg87.18.414.411.97, 1.831.75, 1.70δH 3.27; εNH 7.586.48.214.311.96, 1.851.76, 1.69δH 3.24; εNH 7.59Asn96.78.464.682.84γNH27.54, 6.825.58.294.642.79γNH27.53, 6.82Asp106.78.444.652.807.78.364.752.79Asp117.28.174.622.805.48.354.612.78Ile124.67.843.961.82γH2 1.33, 1.16γH3 0.82; δH 0.694.27.863.971.85γH2 1.34, 1.16γH3 0.83; δH 0.72Phe135.97.744.433.26, 3.10δH 7.32; εH 7.375.5ζH7.317.824.433.25, 3.11δH 7.33; δH 7.37ζH 7.31Arg146.57.754.221.951.76, 1.68δH 3.27; εNH 7.375.67.784.231.931.72, 1.68δH 3.26; εNH 7.37Ile157.07.734.061.97γH2 1.62, 1.22γH3 0.93; δH 0.87Ile166.07.814.121.95γH2 1.53, 1.26γH3 0.96; δH 0.84term-NH2 7.25, 6.91a Numbers in italics, trans andcis-Pro-containing isomers. Open table in a new tab Table IIIStructural statistics of Ni3 and Ci3Ni3Ci3trans-Procis-ProConstraintsIntraresidue213233Sequential876166Medium range561011Total164103110RMSDaRMSD, root mean square deviation from pairwise comparison between all the structures (Å).Backbone0.2–0.8 (residues 3–18)0.3–1.2 (residues 9–15)Heavy atoms0.3–1.6 (residues 3–18)0.5–2.0 (residues 9–15)〈Energy〉bIn Kcal/mol.Leonnard-Jones-van der Waals429188Hydrogen bondscHydrogen bonds were searched using the Measure Hbond facility of INSIGHT and were regarded as present if the following criteria were satisfied simultaneously: 1) the distance between the donor H and the acceptor O was 120 degrees; 3) the hydrogen bond occurred in at least 50% of the energy minimized structures.Donor NHdNH and CO represent backbone atoms.Acceptor COtrans- and cis-ProDonor NHAcceptor COTrp7Tyr3Arg14Ile12Ala9Leu5Ile15Asp11Leu10Ile6Ile16Ile12Lys11Trp7Lys12Lys8Ala13Ala9Glu15Lys11Ile16Lys12Gln17Ala13Lys18Tyr14Asn19Ile16a RMSD, root mean square deviation from pairwise comparison between all the structures (Å).b In Kcal/mol.c Hydrogen bonds were searched using the Measure Hbond facility of INSIGHT and were regarded as present if the following criteria were satisfied simultaneously: 1) the distance between the donor H and the acceptor O was 120 degrees; 3) the hydrogen bond occurred in at least 50% of the energy minimized structures.d NH and CO represent backbone atoms. Open table in a new tab The CD spectra of both peptides did not indicate the existence of any preferential secondary structure in aqueous solution (Fig. 2), and they were not affected by changes in concentration within the 0.15–1.5 mm range. 2Pertinhez, T. A., et al., manuscript in preparation.However, upon addition of small aliquots of TFE, it was found that Ni3 exhibited a transition to an α-helical conformation the extension of which increased up to 30% TFE (Fig. 2 A), where it stabilized at an helical content of ∼55%, as calculated according to the method of Chen et al. (40Chen Y.H. Yang J.T. Chau K.H. Biochemistry. 1974; 13: 3350-3359Crossref PubMed Scopus (1971) Google Scholar). For Ci3, instead, only a partial folding into an ill defined helical structure could be observed at high TFE concentration (Fig. 2 B). In this case, however, due to the complexity of the spectrum suggesting the existence of a multiple conformation equilibrium, the use of the same method to evaluate the peptide α-helical content (Fig. 2 B) appears not to be ideal. A detailed analysis of the CD data will be the subject of a work in preparation.2 According to the CD results, all 1H-NMR experiments were recorded in 70% H2O/30% TFE-d 3 (v/v). The complete assignment of the proton resonances for the two peptides was obtained using standard two-dimensional methods (41Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, New York1986Crossref Google Scholar). The spectra of Ci3 displayed two distinct sets of resonances for the stretch Lys6–Arg14 (TableI), suggesting the existence of acis-trans isomerization about the Lys6—Pro7 peptide bond. The correct assignments of the protons corresponding to the two conformers was based on the observation, in the NOESY and rotating frame nuclear Overhauser effect spectra, of the characteristic connectivities between Lys6αH and Pro7αH for the cisisomers and between Lys6αH and Pro7δH for the trans isomers. The relative intensity of the peaks indicates that the cis-trans molar ratio turns out to be ∼1:1.5, suggesting that the two isomers are energetically quite similar. The chemical shifts for Ci3 and Ni3 are listed in Tables I and II, respectively. The proton resonances of the common sequence EIQKN present significant differences in the two peptides, indicating that the amino acids flanking that domain play an important role in defining its secondary structure. The conformational features of the two peptides have been derived by the analysis of complementary NMR parameters such as the α-proton chemical shift perturbation, the3 J HN-α coupling constants and the NOEs pattern. The α-proton residual chemical shifts (42Wishart D.S. Sykes B.D. Richards F.M. Biochemistry. 1992; 31: 1647-1651Crossref PubMed Scopus (2024) Google Scholar) are reported in Fig.3. In the case of Ni3 (Fig.3 A), it is evident that, except for Ser2 and Leu5, all the α-proton resonances move upfield with respect to the random coil values, thus suggesting the existence of a helical structure extending from Ile6 to Asn19. The presence of sequential d NN (i,i + 1) NOEs, spanning almost the entire Ni3 sequence in the 150-ms NOESY spectrum (Fig.4 A), indeed supports the existence of an α-helical conformation whose stability is confirmed by the characteristic d αN (i,i + 3) connectivities encompassing residues Tyr3–Asn19 (Fig. 4 B). The summary of the interresidue NOEs is reported in Fig.5 A, showing a good correlation with the αCH secondary shifts (Fig. 3 A). Finally, except for Asn19, the majority of the NH-αCH coupling constants (3 J HN-α) are in the range of 5.60–6.5 Hz (Table II), as expected for a peptide in α-helical conformation. On the other hand, knowing that a minimum of 4 adjacent residues with a negative deviation of the αCH chemical shifts is necessary to assess the presence of a stable helical organization, and that its stability is proportional to the intensity of such deviation (42Wishart D.S. Sykes B.D. Richards F.M. Biochemistry. 1992; 31: 1647-1651Crossref PubMed Scopus (2024) Google Scholar), the secondary shifts plot for Ci3 (Fig. 3B) indicates that, in this case, a helical folding is possible only in the peptide COOH-terminal portion. In addition, except for Arg8 and Asp10, all the residues do not exhibit any significant difference between thetrans and the cis isomers (Fig. 3B) suggesting that Ci3, in the two states, adopt very similar conformations. Indeed, the analysis of the NOESY and rotating frame nuclear Overhauser effect spectra carried out for both the trans andcis isomers evidenced nearly no difference. Thus, Fig. 5B shows the summary of the interresidue NOEs only for the relatively more populated trans isomers. As for Ni3, a good correlation with the αCH secondary shifts (Fig. 3B) can be observed. In fact, adjacent NH-NH correlations, together with medium and weak intensitiesd αN (i, i+3) andd αβ (i, i+3) NOEs were found from Asp10 to Ile16, indicating the presence of a helical conformation only in the COOH-terminal portion of the peptide. Moreover, the coexistence of medium and weak intensity (i, i+3) NOEs together with the intense d αN(i, i+1) correlations, suggests the presence of conformational fluctuations in that stretch. The N-terminal half of Ci3, including the first five amino acids that belong to a well defined α-helix in the COOH-terminal half of Ni3, clearly does not adopt any stable secondary structure. Except for the Asn5 NH-Lys6 NH connectivity, the other medium range NOEs associated to the presence of helical regions are unambiguously absent from the spectra. Finally, in the Pro-containing stretch, NOE connectivities reminiscent of the presence of a β-turn were not found. Following the restrained molecular dynamics protocol described under "Materials and Methods," 80 possible three-dimensional structures were calculated for both peptides. After minimization, 20 structures for each peptide were selected to represent their solution conformation. The structural statistics are reported in Table III. Fig. 6 A displays the Ni3 structures superimposed, for the minimum backbone deviation, between residues Tyr3 and Lys18. The existence of a stable and well defined α-helical folding, involving most of the peptide, is also supported by the analysis of the main chain hydrogen bonds (Table III) and of the φ and ψ dihedral angles (data not shown), carried out using the program DSSP (43Kabsch W. Sander C. Biopolymers. 1983; 22: 2577-2637Crossref PubMed Scopus (12421) Google Scholar). In the case of Ci3, Fig. 6 B refers to thetrans-Pro conformers, and the superimposition of the backbone atoms has been made for the Asn9–Ile15 stretch. As previously mentioned, discussing the NMR data, the structures of the cis andtrans isomers are quite similar, with an α-helical conformation extending over the COOH-terminal peptide stretch, only. Because of the limited number of NMR constraints available, the NH2-terminal region preceding Asn9 is poorly defined and no elements of stable ordered secondary structure have been detected. Fig. 6 (bottom) shows a schematic representation of the least energy structure for the two peptides evidencing the amphipathic nature of the α-helical regions. There is a general consensus (6Bourne H.R. Curr. Opin. Cell Biol. 1997; 9: 134-142Crossref PubMed Scopus (530) Google Scholar,44Wess J. FASEB J. 1997; 11: 346-354Crossref PubMed Scopus (511) Google Scholar, 45Strader C.D. Fong T.M. Totá M.R. Underwood D. Dixon R.A.F. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (996) Google Scholar, 46Kobilka K.B. Gether U. Adv. Pharmacol. 1998; 42: 470-473Crossref PubMed Scopus (8) Google Scholar) on the hypothesis that the specific conformational changes of GPCRs cytosolic domains, induced by agonist binding, are critical for their activation. Since experimental observations with substitution or deletion in receptor mutants cannot rule out the induction of indirect conformational effects, the use of synthetic peptides representing defined receptor regions allows the performance of experiments in a more conformationally controlled manner. The validity of this approach is based on the recognition that if the isolated fragments retain the biological function they have in the receptor (e.g.G-protein activation), it is likely that their folding and conformational dynamics are similar to the ones they undergo in the native receptor (33Shirai H. Takahashi K. Katada T. Inagami T. Hypertension. 1995; 25: 726-730Crossref PubMed Google Scholar, 44Wess J. FASEB J. 1997; 11: 346-354Crossref PubMed Scopus (511) Google Scholar). Due to the recognized relevance of the i3 loop as a critical region for GPCRs to express their activity, many attempts are being carried out to identify the domains involved in the complex molecular process of G-protein coupling (6Bourne H.R. Curr. Opin. Cell Biol. 1997; 9: 134-142Crossref PubMed Scopus (530) Google Scholar, 44Wess J. FASEB J. 1997; 11: 346-354Crossref PubMed Scopus (511) Google Scholar, 45Strader C.D. Fong T.M. Totá M.R. Underwood D. Dixon R.A.F. Annu. Rev. 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