Characterization of the bioactive conformation of the C-terminal tripeptide Gly-Leu-Met-NH 2 of substance P using [3-prolinoleucine10]SP analogues Jean Quancard, Philippe Karoyan, Sandrine Sagan, Odile Convert, Solange Lavielle, Ge ´ rard 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-andtrans-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 calcula- tions. From NMR and modeling studies it can be proposed that residue Leu10 of SP adopts a gauche(+) conformation around the v 1 angle and a trans conformation around the v 2 angle in the bioactive conformation. Together with previ- ously published results, our data indicate that the C-terminal SP tripeptide should preferentially adopt an extended con- formation or a PPII helical structure when bound to the receptor. Keywords: substance P; NK-1 receptor; bioactive confor- mation; prolinoleucine. The introduction of a cyclic structure into a polypeptide greatly limits the inherent flexibilities of the peptide backbone and side chains. Disulfide bridging, lactam cyclization and substitution by proline residues are the easiest and therefore the most frequently used strategies. These constraints are introduced to probe the conformation of the substituted residue(s) and/or to generate more specific peptide analogues with high affinities (due to reduction of entropy). Proline, the only ÔnaturalÕ cyclic amino acid, has a restricted F-value of around )60° that constrains the peptide backbone. When inserted into a peptide sequence, a biologically potent proline-substituted analogue of the initial peptide gives information on both the F-value of the substituted residue and the nonimportance of its side chain. If, in contrast, this side chain is mandatory for full biological potency, substituted proline analogues on Cb (position 3), Cc (position 4) or Cd (position 5) may restore the information carried by the side chain. These proline analogues may be used to probe both the orientation of the peptide backbone (/, w) and side chain conformation [v 1 gauche(+) ¼ ) 60°, trans ¼ 180° and gauche(–) ¼ 60°]. Proline has been widely used as a scaffold and substitu- tions on the pyrrolidine ring have yielded a large variety of proline analogues [1–19]. Such constrained templates have been introduced in peptides to elucidate their bioactive conformation: for instance, 3-methylthiomethylproline (3-prolinomethionine) in substance P (SP) [20], 3-(p-hy- droxyphenyl)proline (3-prolinotyrosine) in opioid peptides [21] as well as 3-n-propylproline [22], 3- and 4-alkylthio- prolines [23] in cholecystokinin analogues. Most of cis-andtrans-3-substituted prolinoamino acids (P c 3 aa and P t 3 aa, Fig. 1) bearing a side chain of a natural amino acid can be prepared. These prolinoamino acids chimera may constitute valuable conformational tools assuming that their preferred three-dimensional structures overlap some, if not all, canonical (helical, extended) structures of a-amino acids. Figure 2 shows the dependence of v 1 torsion angle upon the ring pucker and the cis/trans diastereoisomerism. In cis-prolinoamino acids, the Cc-endo ring pucker corresponds to a v 1 around 150° (assimilated to the trans rotamer) and the Cc-exo ring pucker is associated with v 1 around 90° [assimilated to the gauche(–) rotamer]. On the contrary, Cc-endo and Cc-exo ring puckers of trans- prolinoamino acids are related to gauche(+) (v 1  )90°) and trans rotamers (v 1  )150°), respectively. Thus, the Correspondence to O. Lequin, UMR 7613 Paris 6-CNRS, Structure et fonction de mole ´ cules bioactives, Case courrier 45, Universite ´ P. et M. Curie, 4, Place Jussieu, 75252 Paris cedex 05, France. Fax: + 33 1 44 273115, Tel.: + 33 1 44 273843, E-mail: lequin@ccr.jussieu.fr Abbreviations:Boc,(tert-butyloxy)carbonyl; CHO, Chinese hamster ovary; CSD, chemical shift deviation; HBTU, O-benzotriazol-1-yl- N,N,N¢,N¢-tetramethyluronium hexafluorophosphate; IP, inositol phosphate; P 3 aa, prolinoamino acid; PLC, phospholipase C; P c 3 Leu, cis-3-prolinoleucine; P t 3 Leu, trans-3-prolinoleucine; P c 3 Met, cis-3- prolinomethionine; P t 3 Met, trans-3-prolinomethionine; PtdIns, phos- phatidyl inositol; SP, substance P (H-Arg-Pro-Lys-Pro-Gln-Gln- Phe-Phe-Gly-Leu-Met-NH 2 ); SPPS, solid phase peptide synthesis. (Received 26 February 2003, revised 29 April 2003, accepted 13 May 2003) Eur. J. Biochem. 270, 2869–2878 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03665.x cyclic constraint excludes one v 1 gauche rotamer, gauche(+) for cis-prolinoamino acids and gauche(–) for trans-prolinoamino acids, respectively. The C-terminal conformation of SP (H-Arg-Pro-Lys- Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH 2 ) has been probed with proline or prolinoamino acids chimera. The biological activities on the tachykinin NK-1 receptor of [Pro9]SP, [Pro10]SP, [Pro11]SP and both [P c 3 Met11]SP and [P t 3 Met11]SP have been previously reported [20,24]. In the case of prolinomethionine incorporation, we have shown that both [P c 3 Met11]SP and [P t 3 Met11]SP are equipotent to SP, indicating that the v 1 angle of Met11 should be trans. We now report the incorporation of prolinoleucines at position 10 of SP. Indeed, the side chain of Leu10 is critical for NK-1 receptor recognition and agonist activity [25,26]. In contrast to prolinomethionines, only [P t 3 Leu10]SP reta- ins affinity and potency towards the NK-1 receptor. The structural implications of these results on the bioactive conformation of Leu10 and the C-terminal tripeptide of SP are analyzed by NMR spectroscopy on SP analogues and molecular mechanics calculations on model peptides. Experimental procedures Materials [11- 3 H][Pro9]SP (65 CiÆmmol )1 ) was synthesized at Com- missariat a ` l’Energie Atomique (Saclay, France) according to Chassaing et al.[27].[ 3 H]propionyl[Met(O 2 )11]SP(7–11) (95 CiÆmmol )1 ) was synthesized as described previously [28]. Boc-P t 3 Leu and Boc-P c 3 Leu were synthesized as described elsewhere [19]. Peptide synthesis [P c 3 Leu10]SP and [P t 3 Leu10]SP syntheses were carried out on an ABI Model 431 A peptide synthesizer starting from an a-p-methylbenzhydrylamine (MBHA resin, substitution: 0.9 mmolÆg )1 of resin). All Na-Boc-amino acids, in a five- or 10-fold excess, were assembled using N,N¢-dicyclohexylcar- bodiimide and 1-hydroxybenzotriazole as coupling reagents and HBTU for P c 3 Leu and P t 3 Leu. The residues Na-Boc- Met, Na-Boc-P c 3 Leu or Na-Boc-P t 3 Leu, and Na-Boc-Gly were coupled manually to the resin. In order to obtain a lower degree of substitution of the resin, 0.5 mmol of Na-Boc-Met per gram of resin was used for Na-Boc-Met coupling, yielding after acetylation (acetic anhydride/ dichloromethane, 1 : 5), a substitution of around 0.25 mmolÆg )1 of resin for the C-terminal amino acid, as determined by the Gisin test after Boc-deprotection [29]. The syntheses were then carried out on a 0.06-mmol scale. After removal of the last Na-Boc-protecting group, the resin was dried in vacuo. The peptide resin was transferred into the Teflon vessel of an HF apparatus and the peptide was cleaved from the resin by treatment with 1.5 mL of anisole, 0.25 mL of dimethyl sulfide, and 10 mL of anhydrous HF per gram of peptide-resin for 1 h at 0 °C. After lyophiliza- tion of the extract, the crude peptide was purified by preparative reverse phase HPLC. The separation was accomplished using various acetonitrile gradients in aque- ous 0.1% trifluoroacetic acid at a flow rate of 6 mLÆmin )1 with UV detection fixed at 220 nm. Before pooling, the purity of collected fractions was verified by analytical HPLC in isocratic mode at a flow rate of 1.5 mLÆmin )1 with UV detection fixed at 220 nm. Mass spectral analysis was Fig. 1. Schematic representation of 3-prolinoamino acids and descrip- tion of torsion angles. The nomenclature used for proteins side chain rotamers has been adopted. Fig. 2. Structures showing the two ring puckers and the associated side chain conformers of prolinoamino acids. 2870 J. Quancard et al. (Eur. J. Biochem. 270) Ó FEBS 2003 performed by MALDI-TOF. The calculated mass for both [P c 3 Leu10]SP and [P t 3 Leu10]SP is (MH + ) ¼ 1370.70; found mass: [P c 3 Leu10]SP (MH + ) ¼ 1373.76, [P t 3 Leu10]SP (MH + ) ¼ 1373.61. Cell culture CHO cells expressing human NK-1 receptors were cultured in DMEM medium supplemented with 100 IUÆmL )1 peni- cillin, 100 IUÆmL )1 streptomycin, and 10% fetal bovine serum. Cultures were kept at 37 °C in a humidified atmosphereof5%CO 2 . Stable transfections were main- tained by geneticin periodic selection. Binding assays on CHO cells Binding assays were carried out at 22 °C on whole cells in Krebs–Ringer phosphate solution consisting of 120 m M NaCl, 4.8 m M KCl, 1.2 m M CaCl 2 ,1.2m M MgSO 4 ,and 15.6 m M NaH 2 PO 4 , pH 7.2, containing 0.04% bovine serum albumin (w/v), 0.6% glucose (w/v), 1 m M phenyl- methylsulfonyl fluoride, and 1 lgÆmL )1 soybean trypsin inhibitor, as described [28]. Measurements of inositol phosphate and cAMP formation PI hydrolysis and cAMP accumulation were determined as described previously [30]. NMR spectroscopy Lyophilized peptides were dissolved in 550 lL of methanol (C 2 H 3 OH or C 2 H 3 O 2 H) at concentrations around 4 m M . NMR experiments were recorded on Bruker Avance spectrometers at a 1 H frequency of 500 MHz and were processed with XWIN - NMR software, as described previously [31]. Spectra were acquired at temperatures from 278 to 298 K. Solvent suppression was achieved by presaturation during the relaxation delay or with a Watergate sequence [32]. Proton assignments were obtained from the analysis of TOCSY (20 and 95 ms isotropic mixing times) [33] and NOESY experiments (400 ms mixing time) [34]. 3 J HNH a and 3 J H a H b coupling constants were measured on 1D spectra acquired with 16K data points and a spectral width of 5000 Hz. The chemical shift deviations of Ha protons were calculated using random coil values reported in methanol [35]. 1 H assignments of [P c 3 Leu10]SP and [P t 3 Leu10]SP are available as supplementary material. Molecular mechanics calculations Ac-P c 3 Leu-NHMe, Ac-P t 3 Leu-NHMe, Ac-Leu-NHMe, Ac-Gly-Leu-NHMe, Ac-Gly-Pro-NHMe, Ac-Gly-P c 3 Leu- NHMe,Ac-Gly-P t 3 Leu-NHMe,Ac-Leu-Met-NH 2 ,Ac-Leu- Pro-NH 2 ,Ac-Leu-P c 3 Met-NH 2 and Ac-Leu-P t 3 Met-NH 2 were built using InsightII package (Accelrys Inc.). All peptide bonds were fixed in a trans conformation. Mole- cular mechanics calculations were performed with the Discover program and CFF91 forcefield. The electrostatic potential was calculated in vacuo with a distance-dependent dielectric screening of 4Ær. One hundred to 1000 structures were generated by molecular dynamics at 1000 K, saving snapshots every 2 ps. Each structure was then minimized using steepest descent, conjugate gradient and Newton– Raphson algorithms until the gradient was less than 0.001 kcalÆmol )1 Æ A ˚ )1 . NMR structures of SP analogues were calculated in DISCOVER by restrained molecular dynamics as described previously [31]. Results Peptide synthesis of [P 3 Leu10]SP analogues The coupling of the bulky amino acids P c 3 Leu and P t 3 Leu was inefficient using the standard N,N¢-dicyclohexylcarbo- diimide/1-hydroxybenzotriazole procedure and required HBTU as coupling reagent. Consequently, to ascertain the quality of the coupling, the three C-terminal residues were coupled manually. Using this strategy, [P c 3 Leu10]SP and [P t 3 Leu10]SP were obtained with purities and yields similar to SP and [Pro10]SP [36]. Pharmacology of the [P 3 Leu10]SP analogues The affinities of [P 3 Leu10]SP analogues for the two specific binding sites associated with the human NK-1 receptor, NK-1M and NK-1m, have been measured with transfected CHO cells. The more abundant binding site NK-1M (85%) is labeled by 3 H[Pro9]SP and is coupled to cAMP produc- tion, whereas the less abundant binding site NK-1m (15%) is labeled by 3 H-propionyl[Met(O 2 )11]SP(7–11) and is associated with IPs production [20,28]. The binding affinit- ies and agonist potencies of the SP analogues are expressed as K i for NK-1M (major site) and NK-1m (minor site), and EC 50 values for the cAMP and IPs pathways, and are reported in Table 1 [20,28]. The affinity of the trans analogue [P t 3 Leu10]SP is similar to that of SP and [Pro9]SP at both binding sites. The EC 50 values of this analogue to stimulate the cAMP and PLC pathways are in good agreement with the expected theoretical values, calculated from previously established correlations between K i and EC 50 [20]. In contrast, the cis isomer [P c 3 Leu10]SP is a very weak competitor with high K i values for NK-1M and NK-1m specific binding sites. Its efficacy is lower than that of SP on both second messenger responses. [P c 3 Leu10]SP is a partial agonist (pK B 5.12) on the cAMP pathway while no antagonist activity could be detected with this analogue on IPs production. Surprisingly, the K i values of both [P c 3 Leu10]SP and [P t 3 Leu10]SP for NK-1M and NK-1m specific binding sites are in the same range whereas agonists generally have K i values that differ at least by one order of magnitude between the two binding sites. NMR spectroscopy of SP analogues Due to its inherent flexibility, SP is largely unstructured in water but folded conformers are stabilized in lower dielectric constant solvents such as methanol and in lipid environ- ments [31,37,38]. The stabilized conformations in these environments are in agreement with the current state of knowledge on the postulated bioactive conformation of SP [24]. To analyze the structural effects of a P 3 Leu insertion in Ó FEBS 2003 Insertion of prolinoleucines in substance P (Eur. J. Biochem. 270) 2871 the sequence of SP, the conformations of [P c 3 Leu10]SP and [P t 3 Leu10]SP have been studied by NMR spectroscopy in methanol. Cis/trans isomerism. Weak additional resonances could be observed in the spectra of SP analogues. These minor forms involve spin systems in the N-terminus and are likely due to cis/trans isomerism of peptide bonds preceding Pro2 and/or Pro4. However the proline isomerism was not further analyzed, as the proportions of these forms were too weak to give rise to NOE crosspeaks. In all peptides, the major form corresponds to a trans conformation of all peptide bonds, as shown by ad(i)1, i) sequential connectivities for residues Pro2, Pro4, P c 3 Leu10 and P t 3 Leu10. The propor- tion of minor species remains very low (<5%) except in the case of [P c 3 Leu10]SP, for which an additional minor form is more significantly populated ( 15%). The observation of an NOE correlation between Ha Gly9 and Ha P c 3 Leu10 in this minor form demonstrates that the peptide bond between Gly9 and P c 3 Leu10 adopts a cis conformation. Conformational analysis of prolinoamino acid residues. The conformation of the pyrrolidine ring was analysed using the 3 J H a –H b coupling constant and intraresidual NOEs. In P t 3 Leu, the two ring puckers are characterized by divergent 3 J H a –H b coupling constants (10 Hz for the Cc-exo form,  1.5HzfortheCc-endo form, using a Karplus relationship [39]). The 3 J H a –H b coupling constant of P t 3 Leu10 is 7.0 Hz, indicating that there is a conformational equilibrium between the two puckerings. Based on 3 J H a –Hb coupling constant, the proportion of Cc-exo (v 1 trans) conformers can be estimated to be 70% for P t 3 Leu. NMR analysis of [P t 3 Met11]SP gave a 3 J H a –H b coupling constant of 5.2 Hz (unpublished data) which corresponds to 50% of Cc-exo conformers. These data, together with that reported for Ac-trans-3-MePro-NHMe ( 3 J H a –H b 3.2 Hz in chloro- form) [40], suggest that the equilibrium is shifted toward the Cc-exo form (v 1 trans) on increasing the bulkiness of the b-substituent on the pyrrolidine ring (3-MePro <P t 3 Met < P t 3 Leu). In cis-prolinoleucine, the two ring puckers are characterized by close 3 J H a –H b coupling con- stants ( 6–8 Hz), which therefore cannot be used for the conformational analysis. However the two ring puckers differ by their intraresidual Ha–Hc and Hb–Hd NOEs. In [P c 3 Leu10]SP, the prolinoamino acid exhibits a strong Hb-Hd NOE and no Ha–Hc NOE, showing that the Cc-endo form (v 1 trans) predominates. Secondary structure. The chemical shift deviations of Ha protons, which depend on the secondary structure of the peptide backbone [41], are reported in Fig. 3. SP, [P c 3 Leu10]SP and [P t 3 Leu10]SP are characterized by upfield shifts of Ha protons in the 4–7 region, indicative of helical conformations. This helical structure is further supported by the presence of characteristic aN(i, i +3) and aN(i,i+4) medium-range NOEs and small coupling constants for residues Gln5 and Gln6 (<6.3 Hz). The incorporation of a prolinoamino acid in position 10 has no major effect on the Ha CSD of preceding residues. Three-dimensional structure. The structures of the two SP analogues have been calculated by restrained molecular dynamics. They are similar to that of SP in segment 1–7 and are only slightly different in the C-terminus (Fig. 4). Segment 1–3 is found to adopt an extended conformation whereas residues 4–8 form a helical structure. The confor- mations of residues 8–9 are ill defined in the peptides. Thus, the introduction of a prolinoamino acid in the C-terminal tail of SP does not affect the overall structure of the peptide backbone and in particular of the core helical region. Molecular modeling Conformations of prolinoleucine. Minimum-energy con- formations of Ac-P c 3 Leu-NHMe, Ac-P t 3 Leu-NHMe and Ac-Leu-NHMe were generated by molecular mechanics using CFF91 forcefield and results are reported in Tables 2 and 3. The same calculations were performed using AMBER forcefield and yielded similar results, which are not shown here. Table 1. Affinities and activities of cis-andtrans-3-prolinoamino acid-substituted analogues of SP. The affinities of SP and SP analogues were measured for the NK-1M (labeled with 3 H[Pro9]SP) and the NK-1m (labeled with 3 H-propionyl[Met(O 2 )11]SP(7–11) binding sites in CHO cells expressing the human NK-1 receptor, as well as their related potencies to stimulate adenylate cyclase and phospholipase C. The antagonist potency value, pK B , is obtained from pK B ¼ log(DR–1)–log[B].DRisthedose-ratio[A]¢/[A] where [A]¢ is the concentration of agonist A, in the presence of the antagonist B, which is equiactive to [A], the concentration of agonist in the absence of antagonist. Values presented are the mean ± SE of at least three independent experiments run in triplicate. Peptide K i , NK-1M (n M ) EC 50 , adenylate cyclase (n M ) K i , NK-1m (n M ) EC 50 , phospholipase C (n M ) SP a 1.6 ± 0.4 8 ± 2 0.13 ± 0.02 1.0 ± 0.2 [Pro9]SP a 1.1 ± 0.1 10 ± 2 0.13 ± 0.02 0.7 ± 0.1 Propionyl[Met(O 2 ) 11 ]SP(7–11) a 1900 ± 450 >5000 10 ± 2 >5000 [P c 3 Leu10]SP 465 ± 105 >10 000 220 ± 30 260 ± 30 12 ± 3% at 10 )5 M (49 ± 3%) pK B ¼ 5.12 ± 0.06 No antagonism [P t 3 Leu10]SP 0.86 ± 0.07 39 ± 9 0.34 ± 0.09 2.1 ± 0.3 [P c 3 Met11]SP b 2.1 ± 0.1 35 ± 1 0.08 ± 0.005 0.8 ± 0.1 [P t 3 Met11]SP b 3.0 ± 0.5 25 ± 2 0.10 ± 0.007 1.4 ± 0.3 a,b Results already published in [26] and [20], respectively. 2872 J. Quancard et al. (Eur. J. Biochem. 270) Ó FEBS 2003 The relative energies of ring puckers of P c 3 Leu and P t 3 Leu indicate that the isopropyl substituent destabilizes the Cc-exo conformer [v 1 gauche(–)] in Ac-P c 3 Leu-NHMe and the Cc-endo conformer [v 1 gauche(+)] in Ac-P t 3 Leu- NHMe(Tables2and3).Theincreaseoftheenergy difference between the v 1 gauche(–) and trans rotamers is particularly important with Ac-P c 3 Leu-NHMe. Because of the constraint imposed by the pyrrolidine cycle, the / torsion angle of these prolinoamino acids is restrained to values between )95° and )60°,theCc-endo pucker being associated with more negative values of / torsion angle (Tables 2 and 3), as previously reported for proline and proline derivatives [42]. The lower limit values of / ( )90°) are related to minimum-energy conformers corresponding to reverse c-turn, for which the puckering is always Cc-endo. Although structures corresponding to w  )40° (helical) and w  90° (reverse c-turn) are found, the most stable conformations correspond to extended structures with w torsion angle between 110° and 165°.Thew angles of Ac-P t 3 Leu-NHMe are smaller for Cc-exo puckering than for Cc-endo puckering, as observed for proline [42]. The opposite effect is observed for Ac-P c 3 Leu-NHMe, for which the cis isopropyl substituent constrains more the w torsion angle. As previously reported [40], reverse c-turn is desta- bilized for cis-prolinoamino acids due to steric interactions between the b-substituent and the carbonyl oxygen. Low-energy conformations of all model amino acids are shown in (v 1 , v 2 ) maps (Fig. 5). We fixed a limit of 3kcalÆmol )1 to the energy difference (relative to the lowest- energy conformer) for an acceptable structure. Relative energy values of side chain conformations are in agreement with classical (v 1 , v 2 ) maps for Leu in proteins [43]. The conformational space of the prolinoleucines side chain appears to be more limited than that of the parent leucine amino acid. Among the nine (v 1 , v 2 ) conformers of Leu, the most stable conformers are (t, g – )and(g + , t). The three (v 1 , v 2 ) conformers corresponding to a trans v 1 , i.e. (t, t) (t, g – )(t,g + ), are found in both Ac-P c 3 Leu-NHMe and Ac-P t 3 Leu-NHMe. But only one v 1 gauche(+) rotamer is acceptable for Ac-P t 3 Leu-NHMe corresponding to a Fig. 3. Ha chemical shift deviations of SP, [P t 3 Leu10]SP and [P c 3 Leu10]SP analogues. The chemical shift deviations (CSDs) of Ha protons are calculated as the differences between observed chemical shifts and random coil values determined in methanol [35]. The random coil value of Pro was used for the different prolinoamino acids. Therefore, the CSDs of P 3 Leu10 depend primarily on the chemical modification of the pyrrolidine ring and cannot be used for the conformational analysis. Fig. 4. NMR structures of SP [31], [P t 3 Leu10]SP and [P c 3 Leu10]SP analogues in methanol. Residues 4–8 have been used for backbone superposition of the selected conformers. Ó FEBS 2003 Insertion of prolinoleucines in substance P (Eur. J. Biochem. 270) 2873 trans v 2 ,andallv 1 gauche(–) rotamers are energetically excluded in Ac-P c 3 Leu-NHMe. Superimposition of prolinoleucine and leucine. In order to compare the conformational spaces of cis-andtrans- prolinoleucine to that of leucine, minimum-energy struc- tures were systematically superimposed. As expected, only leucine conformations with / torsion angle around )80° (proline-like) fit with prolinoleucines. Although the geometrical constraint due to the cyclization induces a 30° deviation of v 1 from ideal values, all conformers of prolinoleucine acids fit well with the corresponding struc- tures of natural amino acids (rmsd values between 0.024 and 0.06 nm for all heavy atoms). Conformational effects of prolinoamino acids on the preceding residue. Insertion of P t 3 Leu in position 10 and both P c 3 Met and P t 3 Met in position 11 [20] yields potent agonists of NK-1 receptor. In order to analyze the effects of a prolinoamino acid insertion on the backbone Table 2. Relative energies (kcalÆmol -1 )ofminimum-energyconformersofAc-P t 3 Leu-NHMe and corresponding conformers of Ac-Leu-NH 2 that best fit the prolinoamino acid. Conformers highlighted in bold characters have energies beyond 3 kcalÆmol )1 of the global minimum. v 1 ¢, torsion angle is defined by Na,Ca,Cb,Cc¢ atoms of the pyrrolidine cycle. v 1 and v 2 torsion angles correspond to the side chain of the prolinoamino acid and are defined by Na,Ca,Cb,Cc and Ca,Cb,Cc,Cd(proR) atoms. Ac-P t 3 Leu-NHMe Ac-Leu-NHMe DE a w, v 1 , v 2 /wv 1 ¢ v 1 v 2 DE a,t,t -65 -57 -30 -157 177 0.98 2.61 a,t,g – -65 -41 -34 -164 60 0.78 1.29 a,t,g + -64 -54 -32 -161 -70 1.37 2.72 a,g + ,t )76 )22 29 )95 173 3.79 1.71 a,g + ,g – )71 )41 21 )106 61 4.48 2.62 a,g + ,g + )70 )42 18 )107 )68 4.39 2.92 b,t,t -71 112 -29 -157 177 0.14 1.01 b,t,g – -66 147 -33 -163 59 0.00 0.22 b,t,g + -68 118 -31 -161 -68 0.63 1.34 b,g + ,t - 75 162 30 -93 172 2.53 0.69 b,g + ,g – )75 158 25 )102 61 3.41 1.47 b,g + ,g + )74 158 24 )102 )68 3.38 1.76 c,g + ,t - 92 78 32 -91 173 2.80 0.57 c,g + ,g – )91 86 28 )98 60 3.36 1.47 c,g + ,g + )92 86 28 )97 )69 3.38 1.76 a The selected conformers of the amino acid are those that best fit the prolinoamino acid and are not necessarily described by the same torsion angle values. Table 3. Relative energies (kcalÆmol -1 ) of minimum-energy conformers of Ac-P c 3 Leu-NHMe and corresponding conformers of Ac-Leu-NH 2 that best fit the prolinoamino acid. Conformers highlighted in bold characters have energies beyond 3 kcalÆmol )1 of the global minimum. v 1 ¢, torsion angle is defined by Na,Ca,Cb,Cc¢ atoms of the pyrrolidine cycle. v 1 and v 2 torsion angles correspond to the side chain of the prolinoamino acid and are defined by Na,Ca,Cb,Cc and Ca,Cb,Cc,Cd(proR) atoms. Ac-P c 3 Leu-NHMe w, v 1 , v 2 /wv 1 ¢ v 1 v 2 DE Ac-Leu-NHMe DE a a,t,t )73 )37 30 164 161 3.39 2.61 a,t,g – -71 -47 28 159 62 1.29 1.29 a,t,g + -72 -44 30 165 -70 2.92 2.72 a,g – ,t )67 )15 )33 93 170 4.90 3.47 a,g – ,g – )64 )29 )36 87 82 5.72 3.49 a,g – ,g + )64 )21 )34 91 )58 6.79 4.75 b,t,t -74 130 31 166 165 2.27 1.01 b,t,g – -80 111 30 161 57 0.00 0.22 b,t,g + -76 125 31 166 -70 1.71 1.34 b,g – ,t )65 164 )34 92 171 3.41 1.91 b,g – ,g – )62 151 )38 84 82 4.75 2.46 b,g – ,g + )62 157 )36 88 )57 5.49 3.45 a The selected conformers of the amino acid are those that best fit the prolinoamino acid and are not necessarily described by the same torsion angle values. 2874 J. Quancard et al. (Eur. J. Biochem. 270) Ó FEBS 2003 conformation of the C-terminal tripeptide of SP, the minimum-energy conformations of model peptides Ac-Gly-Leu-NHMe, Ac-Gly-Pro-NHMe, Ac-Gly-P c 3 Leu- NHMe, Ac-Gly-P t 3 Leu-NHMe, Ac-Leu-Met-NH 2 ,Ac- Leu-Pro-NH 2 ,Ac-Leu-P c 3 Met-NH 2 and Ac-Leu-P t 3 Met- NH 2 were generated (NHMe and NH 2 C-terminal cappings were chosen to mimic the sequence of SP). Energy differences between minimum structures corresponding to helical (negative w) and extended (positive w) conformations of the first residue are reported in Table 4. The helical conformation of Leu is destabilized when this residue is followed in the sequence by a proline or prolinomethionine and there are no energy minima corresponding to c-turn conformations. These effects can be ascribed to the steric hindrance of the pyrrolidine cycle, as already reported [44,45]. A b-substituent on the pyrrolidine cycle (Met side chain) does not further affect the conformation of leucine. In the case of Gly-Xaa dipeptides, the conformation effect of the pyrrolidine cycle is much smaller on Gly residue and does not significantly restrict its conformational space beside the absence of c-turn minima. Discussion The nonsubstituted proline analogue [Pro11]SP is only a weak competitor of SP binding sites. Reintroducing the methionine side chain in the pyrrolidine ring led to analogues [P c 3 Met11]SP and [P t 3 Met11]SP which are both 400- to 800-times more potent than [Pro11]SP, and therefore equipotent to SP [20]. [Pro10]SP is only slightly less potent than SP (10- to 30-times) [26]. The reintroduction of the leucine side chain restores full potency to [P t 3 Leu10]SP, whereas [P c 3 Leu10]SP is even less potent than [Pro10]SP, being a very weak competitor of SP binding sites. To our knowledge, this is the first time that such a selective recognition is observed between trans and cis-3-substituted prolinoamino acids. Thus, these analogues incorporating the restricted prolinoleucine, with its isopropyl substituent, representedanidealsituationtoascertainthevaluesforthe (/, w, v 1 , v 2 ) torsion angles of leucine in SP, assuming that variations in the structure of SP were mainly restricted to the substituted residue. NMR analysis and molecular modeling studies were performed to reach these torsion angles. Cis-andtrans-3-substituted prolines induce a local constraint restricting the / and v 1 angles of the substituted residue. The v 1 angle is restricted around two values, ± 150° and ± 90° (+ for cis-andfortrans-prolinoamino acids). Our NMR and modeling studies show that the v 1 trans conformers are stabilized on increasing the bulkiness of the b-substituent. The w angle of the prolinoamino acid residue is also restrained. In particular, the introduction of a b-substituent with a cis stereochemistry induces a destabil- ization of reverse c-turn structures in cis-prolinoamino acids [46], due to interactions with the following carboxamide function. In solution, the global three-dimensional struc- tures of SP incorporating either a cis-ortrans-3-substituted proline are quite similar to that of SP except locally around the substituted position. cis/trans isomerism of the Xaa-P 3 aa amide bond is marginally affected by a C3 substitution of the pyrrolidine ring. Indeed, only [P c 3 Leu10]SP in metha- nolic solution showed a slight increase in the concentration of cis amide bond around Gly9-P c 3 Leu10. This is in agreement with previous studies on 3-alkylprolines [46]. This result justifies the trans orientation fixed to the amide bond of the model compounds for molecular mechanics calculations. Minimum-energy conformers of model dipep- tides, including either a proline or a 3-substituted proline, revealed slight conformational disturbances on the pre- ceding (i)1) amino acid, this effect being related to the proline constraint and not to the presence of a b-substituent on the pyrrolidine ring. Thus, cis-andtrans-3-substituted prolinoamino acids chimera constitute valuable tools to probe relationships between the affinity/activity and the conformation of the substituted residue, i.e. all its torsion angles (peptidic backbone: /, w, and side chain conformer v 1 , v 2 ). Differences in receptor affinity of the modified peptide vs. the initial one can be directly related to variations in the structure of the substituted residue. Fig. 5. v 1 –v 2 plots of minimum-energy conformations of Leu, P t 3 Leu and P c 3 Leu. Structures with energies beyond 3 kcalÆmol )1 of the global minimum were excluded. For the sake of clarity, angle values are traced between 0° and 360°, i.e. 360° was added to all negative angle values. Gradual colors were used from blue (most stable conformers) to red (less stable conformers). Table 4. Energy differences between helical (w < 0) and extended (w > 0) conformations of the first residue in model peptides. Peptide DE (kcalÆmol )1 ) Ac-Leu-NHMe 0.77 Ac-Leu-Met-NH 2 0.21 Ac-Leu-Pro-NH 2 3.39 Ac-Leu-P t 3 Met-NH 2 3.31 Ac-Leu-P c 3 Met-NH 2 2.70 Ac-Gly-NHMe 0.57 Ac-Gly-Leu-NHMe 0.05 Ac-Gly-Pro-NHMe 0.55 Ac-Gly-P t 3 Leu-NHMe 0.45 Ac-Gly-P c 3 Leu-NHMe 0.67 Ó FEBS 2003 Insertion of prolinoleucines in substance P (Eur. J. Biochem. 270) 2875 Only the trans isomer ([P t 3 Leu10]SP) is active, with a 600-fold difference in affinity between [P c 3 Leu10]SP and [P t 3 Leu10]SP. Consequently, only conformations specific to P t 3 Leu are liable to mimic the bioactive conformation of Leu10 in SP. Molecular mechanics calculations and NMR studies show that v 1 trans conformations are the most stable in both P c 3 Leu and P t 3 Leu. Moreover, for these conforma- tions, the three v 2 -rotamers have acceptable energies. These results indicate that all v 1 trans conformations of Leu lie at the intersection of the conformational spaces of P c 3 Leu and P t 3 Leu. Thus, a v 1 trans canbeexcludedforthebioactive conformation of Leu10. Furthermore, modeling studies indicate that the only energetically acceptable (v 1 , v 2 ) conformer corresponds to the (g + ,t)rotamer.Figure6 shows the superimposition of P t 3 Leu and Leu in the postulated bioactive conformation. NMR spectroscopy shows that this conformer is observed in solution (propor- tion of 30% in methanol). Therefore, the selective recogni- tion of [P t 3 Leu10]SP vs. [P c 3 Leu10]SP by the NK-1 receptor allows us to access to both v 1 and v 2 angles of the bioactive conformation of Leu10. [Pro10]SP is 10 to 30 times less potent than SP [24]. The reintroduction of the leucine side chain was spectacular in terms of selectivity of cis-substitu- tion vs. trans-substitution of the pyrrolidine ring, but the active analogue [P t 3 Leu10]SP was almost as potent as SP and not a superagonist. One likely explanation might be that the trans-prolinoleucine binds to the NK-1 receptor as a higher-energy conformer (2.5 kcalÆmol )1 ) compared to the corresponding conformer of leucine (0.7 kcalÆmol )1 ). Together, we can conclude that the proline scaffold can orientate both the peptidic backbone and the side chains of Leu10 and Met11 residues in appropriate regions of the space to be recognized by the NK-1 receptor. The torsion angles of leucine are now more precisely defined, but the variations still allowed around the w torsion angles of glycine, leucine and methionine do not allow us to restrict sufficiently the conformational space. Calculations per- formed on Ac-Leu-P c 3 Met-NH 2 and Ac-Leu-P t 3 Met-NH 2 indicate that helical conformations of Leu are less stable than b-conformations, with more than 2 kcalÆmol )1 differ- ence. Previous studies of SP analogues modified in positions 9 and 10 such as N-methylation or introduction of a spirolactam, indicate that these residues should preferen- tially adopt extended conformations [24,31,47]. All these results suggest that the bioactive conformation of the C-terminal tripeptide should be more or less extended instead of helical as stated in some studies from NMR analysis of SP in micellar medium [37,38,48–57]. Three canonical structures correspond to extended structures: poly c-turn, b-strand and PPII helical conformations. Biologic- ally potent compounds obtained by substitution of the residues 9, 10 or 11 with a proline or a prolinoamino acid permit to exclude the poly c-turn structure. In particular, Met11 should not adopt a c-turn conformation as [P c 3 Met11]SP is as potent as SP. Reverse c-turn conforma- tions for Gly9 and Leu10 can also be excluded on the basis of the calculations performed with model dipeptides. Other constrained SP analogues will be necessary to discriminate between a b-strand and a PPII helical conformation of the crucial 9–11 tripeptide, which is necessary for the biological activity of SP. Conclusion The comparison of conformational spaces of cis-andtrans- prolinoamino acid allows one to access to / and v 1 angles. Energy calculations can further restrict w and v 2 torsion angles. In our study, the incorporation of both P c 3 Leu and P t 3 Leuallowedustoaccesstoallthetorsionanglesof Leu10 in the bioactive conformation of SP: /  )60°, w  150°, v 1  )60° and v 2  180°. 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Lee, S., Suh, Y.H., Kim, S. & Kim, Y. (1999) Comparison of the structures of beta amyloid peptide (25–35) and substance P in trifluoroethanol/water solution. J. Biomol. Struct. Dyn. 17, 381–391. 56. Qi, X.F., Zhorov, B.S. & Ananthanarayanan, V.S. (2000) CD, 1 H NMR and molecular modeling studies of the interaction of Ca 2+ with substance P and Ala7-substance P in a non-polar solvent. J. Pept. Sci. 6, 57–83. 57. Auge, S., Bersch, B., Tropis, M. & Milon, A. (2000) Character- ization of substance P–membrane interaction by transferred nuclear Overhauser effect. Biopolymers 54, 297–306. Supplementary material The following material is available from: http://www. blackwellpublishing.com/products/journals/suppmat/EJB/ EJB3665/EJB3665sm.htm Table S1. 1 Hassignmentof[P t 3 Leu10]SP (in methanol, 278 K). Table S2. 1 H assignment of [P c 3 Leu10]SP (in methanol, 278 K). 2878 J. Quancard et al. (Eur. J. Biochem. 270) Ó FEBS 2003 . Characterization of the bioactive conformation of the C-terminal tripeptide Gly-Leu-Met-NH 2 of substance P using [3-prolinoleucine10]SP analogues Jean. yields similar to SP and [Pro10]SP [36]. Pharmacology of the [P 3 Leu10]SP analogues The affinities of [P 3 Leu10]SP analogues for the two specific binding sites