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Residue Leu10 of substance P (SP) is critical for NK-1 receptor recognition and agonist activity. In order to probe the bioactive conformation of this residue,cis-andtrans-3-substituted prolinoleucines were introduced in position 10 of SP. The substituted SP analogues were tested for their affinity to humanNK-1 receptor specific binding sites (NK-1M and NK-1m) and their potency to stimulate adenylate cyclase and phospholipase C in CHO cells transfected with the human NK-1 receptor. | Eur. J. Biochem. 270 2869-2878 2003 FEBS 2003 doi 10.1046 j.1432-1033.2003.03665.x Characterization of the bioactive conformation of the C-terminal tripeptide Gly-Leu-Met-NH2 of substance P using 3-prolinoleucine10 SP analogues Jean Quancard Philippe Karoyan Sandrine Sagan Odile Convert Solange Lavielle Gerard Chassaing and Olivier Lequin UMR 7613 Paris 6-CNRS Universite Pierre et Marie Curie Paris France Residue Leu10 of substance P SP is critical for NK-1 receptor recognition and agonist activity. In order to probe the bioactive conformation of this residue cis- and trans-3-substituted prolinoleucines were introduced in position 10 of SP. The substituted SP analogues were tested for their affinity to human NK-1 receptor specific binding sites NK-1M and NK-1m and their potency to stimulate adenylate cyclase and phospholipase C in CHO cells transfected with the human NK-1 receptor. trans-3-prolinoleucine10 SP retained affinity and potency similar to SP whereas cis-3-prolinoleucine10 SP shows dramatic loss of affinity and potency. To analyze the structural implications of these biological results the conformational preferences of the SP analogues were analyzed by NMR spectroscopy and minimum-energy conformers of Ac-cis-3-prolinoleucine-NHMe Ac-trans-3-prolinoleucine-NHMe and model dipeptides were generated by molecular mechanics calculations. From NMR and modeling studies it can be proposed that residue Leu10 of SP adopts a gauche conformation around the v angle and a trans conformation around the v2 angle in the bioactive conformation. Together with previously published results our data indicate that the C-terminal SP tripeptide should preferentially adopt an extended conformation or a PPII helical structure when bound to the receptor. Keywords substance P NK-1 receptor bioactive conformation prolinoleucine. The introduction of a cyclic structure into a polypeptide greatly limits the inherent flexibilities of the peptide backbone and side chains. Disulfide bridging
