Báo cáo khoa học: Structural function of C-terminal amidation of endomorphin Conformational comparison ofl-selective endomorphin-2 with its C-terminal free acid, studied by 1 H-NMR spectroscopy, molecular calculation, and X-ray crystallography pot

Structural function of C-terminal amidation of endomorphin Conformational comparison of l-selective endomorphin-2 with its C-terminal free acid, studied by 1H-NMR spectroscopy, molecular calculation, and X-ray crystallography Yasuko In1, Katsuhiko Minoura1, Koji Tomoo1, Yusuke Sasaki2, Lawrence H Lazarus3, Yoshio Okada4 and Toshimasa Ishida1 Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka, Japan Department of Biochemistry, Tohoku Pharmaceutical University, Sendai, Japan Medicinal Chemistry Group, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Kobe, Japan Keywords endomorphin-2; C-terminal-deaminated endomorphin-2; NMR; molecular calculation; X-ray crystal analysis Correspondence Y In, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan Fax: +81 72 690 1068 Tel: +81 72 690 1069 E-mail: in@gly.oups.ac.jp (Received 30 June 2005, revised August 2005, accepted 16 August 2005) doi:10.1111/j.1742-4658.2005.04919.x To investigate the structural function of the C-terminal amide group of endomorphin-2 (EM2, H-Tyr-Pro-Phe-Phe-NH2), an endogenous l-opioid receptor ligand, the solution conformations of EM2 and its C-terminal free acid (EM2OH, H-Tyr-Pro-Phe-Phe-OH) in TFE (trifluoroethanol), water (pH 2.7 and 5.2), and aqueous DPC (dodecylphosphocholine) micelles (pH 3.5 and 5.2) were investigated by the combination of 2D 1H-NMR measurement and molecular modelling calculation Both peptides were in equilibrium between the cis and trans rotamers around the Tyr–Pro w bond with population ratios of : to : in dimethyl sulfoxide, TFE and water, whereas they predominantly took the trans rotamer in DPC micelle, except in EM2OH at pH 5.2, which had a trans ⁄ cis rotamer ratio of : Fifty possible 3D conformers were generated for each peptide, taking different electronic states depending on the type of solvent and pH (neutral and monocationic forms for EM2, and zwitterionic and monocation forms for EM2OH) by the dynamical simulated annealing method, under the proton-proton distance constraints derived from the ROE cross-peak intensities These conformers were then roughly classified into four groups of two open [reverse S (rS)- and numerical (n7)-type] and two folded (F1- and F2-type) conformers according to the conformational pattern of the backbone structure Most EM2 conformers in neutral (in TFE) and monocationic (in water and DPC micelles) forms adopted the open structure (mixture of major rS-type and minor n7-type conformers) despite the trans ⁄ cis rotamer form On the other hand, the zwitterionic EM2OH in TFE, water and DPC micelles showed an increased population of F1- and F2-type folded conformers, the population of which varied depending on their electronic state and pH Most of these folded conformers took an F1type structure similar to that stabilized by an intramolecular hydrogen bond of (Tyr1)NH3+ COO–(Phe4), observed in its crystal structure These results show that the substitution of a carboxyl group for the C-terminal Abbreviations EM1, endomorphin-1; EM2, endomorphin-2; EM2OH, C-terminal free acid endomorphin-2; Tic, tetrahydro-3-isoquinoline carboxylic acid; TIPP-NH2, Tyr-Tic-Phe-Phe-NH2; TSP-d4, 2,2,3,3-tetradeuterio-3-(trimethylsilyl)propionic acid sodium salt FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS 5079 Conformational comparison of endomorphin-2 and its C-terminal free acid Y In et al amide group makes the peptide structure more flexible and leads to the ensemble of folded and open conformers The conformational requirement of EM2 for binding to the l-opioid receptor and the structural function of the C-terminal amide group are discussed on the basis of the present conformational features of EM2 and EM2OH and a possible model for binding to the l-opioid receptor, constructed from the template structure of rhodopsin Many bioactive peptides are a-amidated at the C terminus As the deamination of such peptides leads to a considerable loss of bioactivity, the amide group may be important for this [1] However, the structural role of the amide group is still far from being fully understood at present, although this group determines, in part, peptide stability [2,3] To clarify the structural and functional implication of C-terminal a-amidation, we previously investigated the conformational and interaction differences between C-terminal amidated and deamidated (carboxylated) peptides [4–6], assuming that C-terminal amidation is significantly associated with the bioactive conformation of a peptide or its interaction with a receptor N-Terminal amidated endomorphin-1 (EM1, TyrPro-Trp-Phe-NH2) and endomorphin-2 (EM2, TyrPro-Phe-Phe-NH2) are endogenous opioid peptides isolated from the bovine brain and exhibit the highest specificity and affinity for the l-opioid receptor among the endogenous peptides elucidated so far [7] To examine the effect of the C-terminal amidation of these peptides, the binding affinities and bioassays of EM1, EM2 and their C-terminal free acids EM1OH (TyrPro-Trp-Phe-OH) and EM2OH (Tyr-Pro-Phe-Phe-OH) for the l- and d-opioid receptors were measured Deamination of EM1 and EM2 was shown to cause the marked loss of binding affinity and agonist activity of the l-opioid receptor; a similar decrease in activity was observed for morphiceptin (Tyr-Pro-Phe-Pro-NH2) and its C-terminal free acid [8] Furthermore, the d-opioid receptor selectivity of the l-opioid receptorspecific agonist TIPP-NH2 (Tyr-Tic-Phe-Phe-NH2, where Tic ¼ tetrahydro-3-isoquinoline carboxylic acid) was reported to be increased significantly if the C-terminal amide was replaced by a free acid [9] Therefore, differentiation between the l- and d-opioid receptorselective peptides results from the C-terminal region On the other hand, the biological function of naturally occurring opioid peptides could be explained by the ‘message-address concept’ proposed by Schwyzer [10] According to this concept, EM2 could be divided into a message sequence consisting of Tyr-Pro-Phe and an address sequence consisting of Phe-NH2, where the important feature for the opioid activity is the presence 5080 of a cationic amino group and a phenolic group in position 1, a spacing amino acid in position 2, lipophilic and aromatic residues in positions and 4, and C-terminal amidation [3] Using this concept, a comparative conformational study of EM2 and EM2OH would provide useful information on the structural and functional roles of C amidation in forming the EM2 conformation specific for the l-opioid receptor Therefore, we previously compared the conformations of EM2 and EM2OH in dimethyl sulfoxide, as determined by 1H-NMR spectroscopy and molecular energy calculations, and reported [6] that: (a) substitution of a carboxyl group for the C-terminal amide group makes the molecular conformation of EM2 flexible; and (b) the stable conformation of EM2OH is not compatible with the bioactive l-opioid receptor-selective conformation proposed for EM2 This result appears to be important, because it means that C-terminal amidation, which shifts the N-terminal amino group to a neutral state, participates in forming a defined bioactive conformation To confirm whether this phenomenon is commonly observed in different environments, we have investigated the solution conformations of EM2 and EM2OH in trifluoroethanol (TFE), water (pH 2.7 and 5.2) and aqueous dodecylphosphocholine (DPC) micelles (pH 3.5 and 5.2); some of the results have been reported in the proceedings of the Japanese Peptide Symposium [11] Because the conformation of a biomolecule is largely influenced by the properties of the solvent, such as polarity and dielectric constant, the conformational data measured in these different solutions, together with those in dimethyl sulfoxide [6], will provide reliable and systematic information on the intrinsic conformational features of EM2 and EM2OH, which is important when considering the substrate specificity of l-opioid receptors and the structural role of C-terminal amidation Results Opioid activity The binding affinities of EM1, EM2, EM1OH and EM2OH for l- and d-opioid receptors and the FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS Y In et al Conformational comparison of endomorphin-2 and its C-terminal free acid Table Binding affinities and the pharmacological activities of EM1, EM2, EM1OH and EM2OH for l- and d-opioid receptors Receptor binding In vitro agonist bioassay Compound l-Opioid receptor Ki (nM) d-Opioid receptor Ki (nM) Guinea pig ileum assay (l-opioid receptor) IC50 (nM) Mouse vas deferens assay (d-opioid receptor) IC50 (nM) EM1 EM1OH EM2 EM2OH 0.36 200 0.69 200 1500 1800 9200 3950 10.1 ± 1.2 4032 ± 4330 5.79 ± 0.4 > 104 36.3 ± 5.2 > 104 344 ± 93 > 104 pharmacological activities of these compounds as l- and d-opioid receptor agonists are given in Table Although the bioassays of these peptides by Al-Khrasani et al [12] found only slightly lower (2.3–4.4 times) potencies for EM1OH and EM2OH than those of the parent amides, our results indicated that the deaminations of EM1 and EM2 cause drastic loss of binding affinity and agonist activity for the l-opioid receptor; a similar decrease of activity has been observed for morphiceptin (Tyr-Pro-PhePro-NH2) and its C-terminal free acid [8] The Ki values for the binding affinity also suggest that the d-opioid receptor affinity of EM2 is increased by the substitution of a carboxyl group for the C-terminal amide group, and a similar phenomenon has been reported by Schiller et al [9], where the d-opioid receptor selectivity of the l-opioid receptor-specific (Tyr-Tic-Phe-Phe-NH2) was agonist TIPP-NH2 increased significantly if the C-terminal amide was replaced by a free acid It is obvious from the present results that the differentiation between the l- and d-opioid receptor selectivities of EM2 is related to C-terminal amidation Solution conformation by NMR spectroscopy and simulated annealing calculation The conformational features of EM2 and EM2OH, obtained by the present NMR measurements and molecular modeling calculations are summarized in Table H-NMR spectroscopy Proton peak assignments were performed using a combination of connectivity information via scalar coupling in phase-sensitive TOCSY experiments and sequential ROE networks along peptide backbone protons The high degree of overlap for Phe3 and Phe4 in TFE made unambiguous assignments difficult for these aromatic protons Because of the FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS broad peaks or their extensive overlapping or the fast H–D exchange with the solvent, accurate and complete assignments were not possible for some protons The existence of cis and trans rotamers around the Tyr-Pro amide bond was identified by the ROE observations between Tyr CaH proton and Pro CaH ⁄ CdH protons, and the population ratio determined by the comparison of the proton peak intensities is given in Table The N-terminal amino protons of EM2 and EM2OH, as well as the C-terminal carboxyl proton of EM2OH, were not detected in all of the solutions, probably due to the fast H–D exchange; consequently, it was impossible to determine the electric states of the N-terminal amino groups (cationic or neutral) of EM2 and EM2OH and that of the C-terminal carboxyl group (anionic or neutral) of EM2OH Therefore, EM2 was considered to be in neutral form in TFE and in monocationic form in water and DPC micelles, because the pKa of Tyr is 2.2 Similarly, EM2OH was considered to be in zwitterionic form in TFE, water (pH 2.7 and 5.2) and DPC micelles (pH 3.5 and 5.2), and in monocationic form in water (pH 2.7) A typical difference between the EM2 and EM2OH was observed for the pH dependence of their NMR spectra Characteristically, the NMR spectra of EM2 in water of pH 2.7 and DPC micelles of pH 3.5 were the same as those in solutions of pH 5.2 This was in contrast with the case of EM2OH, where the NMR spectra differed considerably depending on pH The chemical shift changes of NH or OH protons were measured as functions of temperature, and their temperature coefficients are given in Table 3; the temperature coefficients of EM2 protons in water and DPC micelles were hardly influenced by a change in pH Because the temperature coefficients were not measured for all N-terminal amino protons and some C-terminal amide or OH protons, it was impossible to 5081 Conformational comparison of endomorphin-2 and its C-terminal free acid Y In et al Table Summary of the overall conformational characteristics of EM2 and EM2OH in DMSO, TFE, H2O (pH 2.7 and 5.2) and DPC micelles (pH 3.5 and 5.2) Open and fold represent the conformations rS and n7 in parentheses indicate the reverse S- and numerical seven-like-open conformations F1 and F2 represent the folded conformations in which hydrogen bonds are formed and not formed, between the N- and C-terminal polar atoms, respectively The numbers following these symbols indicate the number of conformers that belong to the respective categories from a total of 30 conformers H2O DPC Solvent electronic form pH 2.7 pH 5.2 pH 3.5 pH 5.2 Neutral trans ⁄ cis ¼ : Open (rS ¼ 30) trans ⁄ cis ¼ : – – – trans – – trans ⁄ cis ¼ : Open (rS ¼ 18, n7 ¼ 4) fold (F2 ¼ 8) – Neutral Open (rS ¼ 30) Monocation cis TEF Monocation EM2 trans DMSO – Open (rS ¼ 13, n7 ¼ 7) fold (F2 ¼ 10) – Monocation cis trans ⁄ cis ¼ : Open (rS ¼ 6, n7 ¼ 12) fold (F1 ¼ 7, F2 ¼ 5) – – Zwitter Fold (F1 ¼ 30) Fold (F1 ¼ 30) Monocation EM2OH trans – – Zwitter trans ⁄ cis ¼ : Open (n7 ¼ 11) fold (F1 ¼ 14, F2 ¼ 5) Open (rS ¼ 18, n7 ¼ 12) – 5082 – Open (rS ¼ 7, n7 (F2 ¼ 2) trans ⁄ cis ¼ : Open (rS ¼ 4, n7 ¼ 14) fold (F1 ¼ 8, F2 ¼ 4) Open (rS ¼ 18, n7 ¼ 10) fold (F2 ¼ 2) Open (rS ¼ 16, n7 ¼ 2) fold (F1 ¼ 12) Open (rS ¼ 30) draw any conclusions on the conformational feature However, most of the temperature coefficients of NH protons (Dd ⁄ DT ¼ 3.0–9.8 p.p.b.ỈK)1) were not sufficiently small to support the presence of inter- or intramolecular hydrogen bonds in all solvent systems, because a proton with a Dd ⁄ DT coefficient of less than 1.0 p.p.b.ỈK)1 is generally considered as participating in a hydrogen bond [13,14] An exception was observed for one of the two C-terminal amide protons of trans EM2 in water (0.6 p.p.b.ỈK)1), suggesting the participation of this group in any specific interactions (see later discussion) To estimate proton–proton distance, ROESY spectra were measured according to the short-, mediumand long-range ROE connectivities along the peptide backbone Some selected inter-residual ROE connectivities, which show the characteristic differences between the EM2 and EM2OH, are listed in Table As the long-range ROEs, which have a strong influence on determining the overall molecular conformation, were very few, the peptides would be an ensemble of many different conformers However, Open (rS ¼ 17, n7 ¼ 4) fold (F1 ¼ 1, F2 ¼ 8) – – ¼ 20) – trans ⁄ cis ¼ : Open (rS ¼ 21, n7 ¼ 2) fold (F2 ¼ 7) – Open (rS ¼ 5, n7 ¼ 7) fold (F1 ¼ 18) – – trans Open (rS ¼ 12, n7 ¼ 9) fold (F1 ¼ 1, F2 ¼ 8) – trans ⁄ cis ¼ : Fold (F1 ¼ 30) – Fold (F1 ¼ 30) – – – some conformational features could be estimated by taking the possible combination of these inter-residual ROE pairs into consideration 3D molecular construction by simulated annealing calculation Possible 3D structures of EM2 and EM2OH were constructed by the dynamical simulated annealing method using the proton–proton distance constraints derived from the ROE cross peaks: EM2 had 25 ⁄ 22 and 50 ⁄ 30 constraints for the trans ⁄ cis rotamers in TFE and water (pH 2.7 and 5.2), respectively, and 48 constraints for the trans rotamer in DPC micelles (pH 3.5 and 5.2); EM2OH had 22 ⁄ 29, 40 ⁄ 35, 51 ⁄ 50, and 56 ⁄ 37 constraints for the trans ⁄ cis rotamers in TFE, water (pH 2.7), water (pH 5.2) and DPC micelles (pH 5.2), respectively, and 43 constraints for the trans rotamer in DPC micelles (pH 3.5) Also the constraints were imposed for three x torsion angles with an allowance of ± 10° According to solvent type and pH, two types of electronic state were FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS Y In et al Conformational comparison of endomorphin-2 and its C-terminal free acid Table Temperature coefficients (Dd ⁄ DT, p.p.b.ỈK)1) of chemical shift changes of NH and OH protons Tyr1 NH and OH protons (EM2 and EM2OH) and Phe4OH proton (EM2OH) were not observed (–) H2O Residue EM2 trans Phe3NH Phe4NH C-term.NH2 cis Phe3NH Phe4NH C-term.NH2 EM2OH trans Phe3NH Phe4NH cis Phe3NH Phe4NH Conformational characteristics of EM2 DPC TFE pH 2.7 pH 5.2 pH 3.5 pH 5.2 4.06 3.32 3.40 4.29 9.76 6.68 0.60 3.90 9.76 6.68 0.60 3.90 8.47 7.32 – – 8.47 7.32 _ – 4.58 4.20 3.01 5.11 7.70 7.62 – – 7.70 7.62 – – 3.27 5.68 9.49 6.35 8.63 3.00 7.13 6.56 7.67 4.46 3.93 6.10 7.81 7.34 7.18 4.88 6.47 2.01 considered: for EM2 in neutral form (N-terminal NH2 and C-terminal NH2) in TFE and in monocationic form (N-terminal NH3+ and C-terminal NH2) in water and DPC micelles; for EM2OH in zwitterionic form (N-terminal NH3+ and C-terminal COO–) in TFE, water and DPC micelles and in monocationic form (N-terminal NH3+ and C-terminal COOH) in water (pH 2.7) (see Table 2) Starting with 50 different conformation sets with random arrays of atoms, energy-minimization trials were performed to eliminate any possible source of initial bias in the folding pathway, in which the target function was minimized by changing /, w, x and v torsion angles Although neither of the peptides produced wellrefined conformers that agreed perfectly with all constraints imposed in the model, the constructed NMR conformers satisfied the distance constraints within the allowable range and either of four possible / torsion angles (calculated from the coupling constants) within ± 30° On the basis of their backbone conformations, the respective conformers were classified into four groups The open conformers were divided into two groups of ‘numerical (n7)’-like and ‘reverse S (rS)’-like curves; and the fold conformers were divided into two groups according to the interaction pattern between the C- and N-terminal polar atoms, that is, their hydrogen-bonded FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS (F1-type) and nonhydrogen-bonded (F2-type) foldings The results are given in Table As shown in Table 2, EM2 has a trans ⁄ cis rotamer ratio of about : in TFE and water (pH 2.7 and 5.2) The predominance of the trans rotamer has also been observed in dimethly sulfoxide (trans ⁄ cis ratio ¼ : 1) [5] Characteristically, the conformers of EM2 in water and DPC micelles were hardly influenced by the variation in pH, because the NMR spectra of EM2 in a solution of acidic pH were identical to those in solution of pH 5.2 This is in contrast with the EM2OH conformers, whose NMR spectra differed considerably depending on pH (discussed later) A characteristic feature of most conformers of EM2 in DPC micelles was the trans rotamer in both acidic and neutral conditions As EM1 has a trans ⁄ cis equilibrium of ratio 74 : 26 in SDS micelles and the cis rotamer is predominant in reverse AOT (bis(2-ethylhexyl)sulfosuccinate sodium salt) micelles [15], the predominance of the trans rotamer may be dependent on the property of the DPC detergent Trans EM2 As the pK1 of Tyr is 2.2, most EM2 conformers are thought to overwhelmingly take the monocationic electronic form in an aqueous or DPC micelle solution of both acidic and neutral pH, and the neutral form in TFE Most conformers of the trans rotamer converged into the open conformation of the extended backbone structure, twisting at the Pro2-Phe3 moiety Many ROEs between neighbouring residues and minor ROEs between residues separated by more than one residue resulted in the absence of a well-defined overall structure, and the lack of direct ROEs among the aromatic protons of Tyr1, Phe3 and Phe4 leads to the fluctuation of these rings The superimposed backbone structures of 30 energetically stable conformers in the respective solutions are shown in Fig 1, and the most stable conformers that belong to the respective conformational groups are shown in Fig As shown in Fig and Table 2, trans EM2 prefers to form the rS-type open conformers in all solutions, and their main stabilizing factors are the double hydrogen bonds of (Tyr1)C ¼ O HN(Phe3) and (Pro2)C ¼ O HN(Phe4) pairs (Fig 2a), although the Dd ⁄ DT values suggest the other many conformers Table also shows that the flexibility of the overall conformation increases in the various solutions in the order of dimethylsulfoxide < TFE ¼ water < DPC micelles The 5083 Conformational comparison of endomorphin-2 and its C-terminal free acid Y In et al Table Inter-residual ROE pairs and intensities of showing notable difference between EM2 and EM2OH in TFE, water and DPC micelles ROE intensities of neighbouring protons on the same aromatic ring or geminal protons were omitted ROE intensities are classified as weak ˚ ˚ ˚ (1.6 to 5.0 A), medium (1.6 to < 3.5 A), and strong (1.6 to 2.6 A) Proton i TFE trans EM2 cis EM2 H2O (pH 2.7) trans EM2 cis EM2 H2O (pH 5.2) trans EM2 cis EM2 DPC (pH 3.5) trans EM2 DPC (pH 5.2) trans EM2 5084 Proton j Intensity Proton i Proton j Intensity Tyr1 b2 Phe4 a Phe4 a Pro2 d1 C-NH2 C-NH2 Weak Medium Medium trans EM2OH Tyr1 b2 Phe3 NH Weak cis EM2OH Tyr1 a Tyr1 2,6H Tyr1 3,5H Phe3 b2 Phe3 NH Pro2 d1 Pro2 a Phe4 NH Weak Weak Weak Weak Tyr1 b1 Phe3 3,5H Phe4 a Phe4 a Tyr1 b1 Tyr1 b2 Pro2 c2 Pro2 d2 Phe4 a C-NH1 C-NH2 Pro2 a Pro2 a Phe3 NH Weak Weak Medium Weak Medium Strong Weak trans EM2OH Tyr1 2,6H Phe3 2,6H Pro2 d2 Phe4 NH Weak Medium cis EM2OH Tyr1 b2 Tyr1 2,6H Pro2 a Phe3 a Phe3 b2 Phe3 2,6H Pro2 d1 Phe4 NH Phe4 NH Phe4 NH Medium Weak Medium Strong Medium Tyr1 b1 Phe3 3,5H Phe3 b1 Phe4 a Phe4 a Pro2 b2 Pro2 c2 Pro2 d2 Phe4 a Phe4 NH C-NH1 C-NH2 Phe3 NH Phe3 NH Weak Weak Weak Medium Weak Weak Weak trans EM2OH Tyr1 3,5H Pro2 b1 Phe3 NH Pro2 c Phe4 NH Phe4 NH Medium Weak Weak cis EM2OH Tyr1 3,5H Pro2 b1 Phe3 NH Phe3 a Phe3 b2 Pro2 a Phe4 NH Phe4 NH Phe4 NH Phe4 NH Medium Weak Weak Medium Medium Tyr1 2,6H Pro2 b1 Phe3 b2 Phe3 2,6H Phe3 2,6H Phe3 3,5H Phe3 3,5H Phe3 Phe3 Phe4 Phe4 Phe4 Phe4 Phe4 a 2,6H 2,6H 2,6H 3,5H a 2,6H Weak Weak Weak Medium Medium Medium Medium trans EM2OH Pro2 b1 Pro2 b1 Pro2 c1 Pro2 c1 Phe3 a Phe3 a Phe3 b2 Phe4 Phe4 Phe4 Phe4 Phe4 Phe4 Phe4 2,6H 3,5H 2,6H 3,5H b2 2,6H NH Weak Weak Weak Weak Weak Medium Weak Tyr1 2,6H Phe3 2,6H Phe3 2,6H Phe3 2,6H Phe3 3,5H Phe3 3,5H Phe3 Phe4 Phe4 Phe4 Phe4 Phe4 a a 3,5H 2,6H a 2,6H Weak Weak Medium Medium Medium Medium trans EM2OH Pro2 b1 Pro2 c1 Phe3 b1 Phe3 b2 Phe3 Phe3 Phe4 Phe4 NH 2,6H 2,6H NH Weak Weak Weak Weak cis EM2OH Tyr1 a Tyr1 2,6H Tyr1 2,6H Tyr1 3,5H Pro2 a Phe3 NH Phe3 a Phe3 b1 Phe3 b2 Pro2 a Pro2 a Pro2 d1 Pro2 a Phe3 NH Phe4 NH Phe4 NH Phe4 2,6H Phe4 2,6H Medium Medium Weak Medium Medium Weak Weak Medium Weak FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS Y In et al Conformational comparison of endomorphin-2 and its C-terminal free acid A B C Cis EM2 The cis rotamer of EM2 exists in TFE and water, but not in DPC micelles The superimposed backbone structures of 30 energetically stable conformers in TFE and water are shown in Fig 3, and the most stable conformers that belong to the respective conformational groups are shown in Fig The cis EM2 conformers in water are an ensemble of open and folded forms, although the n7-type open form exists as the major conformer in water On the other hand, the conformers in TFE have an increased proportion of the F2-type folded and rS-type open forms, which is mostly due to the hydrophobic interactions among aromatic rings, particularly between Tyr1 and Phe3 aromatic rings It is noteworthy that this F2-type folded conformer (Fig 4D) is similar to the stable form of cis EM1 proposed by Podlogar et al [16], where the molecule adopts a conformation in which the aromatic rings of Tyr1 and Trp3 are packed against the Pro2 ring As a whole, these findings suggest that the conformation of cis EM2 is more flexible than that of trans EM2 in TFE and water, although EM2 in dimethyl sulfoxide solution still takes a welldefined rS-type open conformation despite the difference of cis and trans rotamers In conclusion, this study showed that the solution conformation of EM2 could be grouped into four conformers, that is, F1- and F2-type folded conformers and n7- and rS-type open conformers Although all these conformations are in the minimum energy region, the barrier appears to be sufficiently low to allow reversible conformational transition among them The F1-type folded and rS-type open conformations may be located at both termini of the conformational transition, and the F2-type folded and n7-type open conformations are situated at intermediate positions: F1-type folded form $ F2-type folded form $ n7-type open form $ rS-type open form The population ratio of these four conformers depends on environmental conditions, such as pH, solvent type and temperature Fig Stereoscopic superimpositions of backbone structures of 30 energetically stable conformers of trans EM2 in (A) TFE, (B) water (pHs 2.7 and 5.2) and (C) DPC micelles (pH 3.5 and 5.2) The conformations are overlaid so as to superimpose their Tyr-Pro backbone chains F1-folded conformers exist in DPC micelles, although their population is minor, indicating that the EM2 conformation is relatively easy to transform in this membrane-mimetic circumstance, as compared to DMSO, TFE or water FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS Conformational characteristics of EM2OH The major electronic state of EM2OH could be the zwitterionic form in TFE, water and DPC micelles, and the monocationic form would also exist as a minor form in water of pH 2.7 The trans ⁄ cis rotamer was observed with population ratios of : to : in TFE, water and neutral DPC micelles (pH 5.2), and characteristically EM2OH in DPC micelles of pH 3.5 5085 Conformational comparison of endomorphin-2 and its C-terminal free acid Y In et al Fig Stereoscopic views of most stable conformers of trans EM2 belonging to respective conformational groups (A) rS-type open conformer in TFE, (B) n7-type open conformer in water, (C) F1-type and (D) F2-type folded conformers in DPC micelles existed almost completely as a trans rotamer, similar to the case of trans EM2 Trans EM2OH The superimposed backbone structures of 30 energetically stable conformers of zwitterionic trans EM2OH in TFE, water and DPC micelles are shown in Fig 5; the many stable conformers belonging to the respective conformational groups are almost the same as those of trans EM2 shown in Fig In contrast with EM2, EM2OH consisted of an ensemble of many open and folded conformers, whose population ratio was largely dependent on the electronic state and pH As shown in Table 2, the ensemble of open and folded conformers existed in DMSO, TFE, water (pH 2.7) and DPC micelles (pH 3.5) On the other hand, most EM2OH conformers in water (pH 5.2) showed the rS-type open structures; this is in contrast with the case in DPC micelles of pH 5.2, where all conformers showed the F1-type folded structure stabilized by a (Tyr1)NH O¼C (C-terminal carboxyl) hydrogen bond, as in Fig 2C As this well-defined folded structure was also observed in conformers of cis EM2OH (discussed later), DPC micelles at a neutral pH may shift the conformation of 5086 EM2OH to a folded form despite the difference of trans ⁄ cis rotamer This is in contrast with the case of EM2, where open conformers were preferentially formed despite the difference of pHs On the other hand, the monocationic form, which is possible in water of pH 2.7, preferentially shifted the conformation of trans EM2OH toward the open structure, and this is due to the disappearance of the electrostatic interactions between the cationic N-terminal and anionic C-terminal groups Cis EM2OH The superimposed backbone structures of 30 energetically stable conformers of zwitterionic cis EM2OH in TFE, water and DPC micelles are shown in Fig 6; many stable conformers belonging to the respective conformational groups are almost identical to those of cis EM2 shown in Fig In DPC micelles, the cis rotamer of EM2OH existed only in neutral (pH 5.2) with a trans ⁄ cis ratio of : As is obvious from Table 2, most conformers of cis EM2OH in all solutions preferred to take the F1-type folded conformation through the NH O hydrogen bond between the N- and C-terminal ends, although the equilibrium with FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS Y In et al Conformational comparison of endomorphin-2 and its C-terminal free acid A B Fig Stereoscopic superimpositions of backbone structures of 30 energetically stable conformers of cis EM2 in (A) TFE and (B) water (pH 2.7 and 5.2) The conformations are overlaid so as to superimpose their Tyr-Pro backbone chains rS-type open conformers was formed in water On the other hand, the monocationic form of cis EM2OH in acidic water of pH 2.7 shifted all conformers to the rS-type open form, similarly to the case of trans EM2OH Crystal structure of EM2OH The EM2OH crystal consists of two independent conformers (conformers A and B) and seven water molecules per asymmetric unit These conformers are shown in Fig Selected conformational torsion angles, hydrogen bonds and electrostatic short contacts are given in Tables and Both conformers take the zwitterionic form of the cis configuration around the Tyr1-Pro2 amide bond, where the backbone structure is folded at residues Pro2 and Phe3 Conformer A, which belongs to the F1-type folded conformation, is FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS mainly stabilized by an intramolecular (Tyr1)NH3+ OOC(Phe4) hydrogen bond, in addition to the electrostatic interaction of the (Pro2)N NH(Phe3) atomic pair A water molecule (O2W) is bifurcately hydrogenbonded to the two NH protons of Phe3 and Phe4 residues, playing an auxiliary role in stabilizing this F1 conformation On the other hand, such an intramolecular hydrogen bond was not formed in conformer B However, the conformation itself is very similar to conformer A, except for the Phe3w and Phe4/ torsion angles The folded conformation of conformer B is mainly stabilized by the triple hydrogen bonds of a water molecule (O6W) with NH3+(Tyr1), NH(Phe3) and –OOC(Phe4), in addition to the indirect interaction between both terminal polar groups via a water molecule (O7W): a O7W NH3+(Tyr1) electrostatic interaction and a O7W OOC– (Phe4) hydrogen bond – 5087 Conformational comparison of endomorphin-2 and its C-terminal free acid Fig Stereoscopic views of most stable conformers of cis EM2 belonging to respective conformational groups (A) rS-type open conformer in TFE, (B) n7-type open conformer in water, (C) F1-type folded conformer in water and (D) F2-type folded conformer in TFE Discussion Conformational difference between EM2 and EM2OH: Effect of C-terminal amidation NMR analyses indicated that both EM2 and EM2OH are in equilibrium between open and folded conformers with trans ⁄ cis population ratios of : to : in dimethyl sulfoxide, TFE and water, although the frequency of taking the cis rotamer of EM2OH is higher than that of EM2 In contrast, EM2 takes only the 5088 Y In et al trans rotamer in DPC micelles despite the difference of pH; this is not the case for EM2OH Concerning the temperature dependence of the chemical shifts of Phe3 and Phe4 NH protons, no notable difference was observed between EM2 and EM2OH, indicating that the conformational behaviour of aromatic residues of EM2 is hardly affected by the substitution of C-terminal carboxyl group However, one of two C-terminal amide protons of EM2 in water showed the situation shielded from the effect of solvent This may be because many rS-type open conformers of EM2 form the intramolecular hydrogen bond (C-terminal)NH O ¼ C (Phe3 or Phe4) The substitution of the carboxyl group for the C-terminal amide group increased the population of folded conformer in the molecular conformation, which largely resulted from the change in the electronic state in the solvent, that is, neutral form (in dimethyl sulfoxide and TFE) and monocationic form (in water and DPC micelles) for EM2, and zwitterionic form (in dimethyl sulfoxide, TFE, water, DPC micelles) and monocationic form (in acidic water) for EM2OH Although many conformers of trans EM2 converge into the relatively well-refined open conformation, particularly of the rS-type, those of trans EM2OH are roughly separated into two groups, i.e the open conformers of n7- or rS-type backbone structure and the F1-type folded conformation turned at the Pro2–Phe3 moiety A characteristic feature of the trans EM2OH conformation is that most conformers in water of pH 5.2 take the rS-type open conformation predominantly, whereas all conformers in DPC micelles of the same pH take the F1-type folded conformation The conformational difference was more clearly observed between the cis rotamers of EM2 and EM2OH Neutral cis EM2 in dimethyl sulfoxide or TFE could be converged into an extended open conformation similarly to its trans rotamer, except the orientation of the Tyr1 residue with respect to the Pro2 residue, and monocationic cis EM2 in water also takes the open conformation predominantly In contrast, the conformers of zwitterionic cis EM2OH in dimethyl sulfoxide, TFE or DPC micelles (pH 5.2) overwhelmingly converge into the folded conformation turned at the Pro2–Phe3 sequence, although those in water show conformational variation between the folded and open structures, and a decrease in pH (a monocationic form is possible in water of pH 2.7) increases the population of open conformation The present study demonstrates that conformers of EM2 prefer to take the open conformation The rS-type open conformer of trans EM2, such as that in Fig 2A, exists as the major conformer in all solutions, FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS Y In et al Conformational comparison of endomorphin-2 and its C-terminal free acid A B C D E F Fig Stereoscopic superimpositions of backbone structures of 30 energetically stable conformers of zwitterionic trans EM2OH in (A) TFE, (B) water (pH 2.7), (C) water (pH 5.2), (D) DPC micelles (pH 3.5), (E) DPC micelles (pH 5.2), and of monocationic trans EM2OH (F) in water (pH 2.7) The conformations are overlaid so as to superimpose their Tyr-Pro backbone chains and the cis rotamer could also be classified with the same conformational preference In the case of EM2OH, however, the energy-minimized conformers of the trans rotamer in dimethyl sulfoxide, TFE or water converge into an ensemble of open and folded conformations This resulted from the difference between the C-terminal amide and carboxyl groups and indicates that the substitution of a carboxyl group for a C-terminal amide group makes more easy the transition between the folded and open conformers of EM2 The C-terminal carboxylic acid allows formation of the folded conformation turned at the Pro2–Phe3 sequence, especially for the cis rotamer, because the ionic interaction (including an intramolecular hydrogen FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS bond) between the N-terminal NH3+ and C-terminal COO– groups acts as a driving force for folding the molecular conformation In other words, the C-terminal amide group plays a role in preventing the formation of such a folded conformation Single crystals of EM2OH were successfully obtained from neutral water adjusted to pH 5.2 by NaOH addition, whereas the crystals were not obtained from water (pH 2.3) The crystal analysis showed two independent, but similar F1-type folded conformers of zwitterionic cis EM2OH, which also existed in dimethyl sulfoxide, TFE, water, or DPC micelles, indicating that this F1-type folded conformer is one of the most stable conformers of EM2OH 5089 Conformational comparison of endomorphin-2 and its C-terminal free acid Y In et al Fig Stereoscopic superimpositions of backbone structures of 30 energetically stable conformers of zwitterionic cis EM2OH in (A) TFE, (B) water (pH 2.7), (C) water (pH 5.2), (D) DPC micelles (pH 5.2), and of monocationic cis EM2OH (E) in water (pH 2.7) The conformations are overlaid so as to superimpose their Tyr-Pro backbone chains Conformational comparison between cis and trans rotamers of EM2 The present study indicated that many conformers of cis and trans EM2 rotamers prefer to take the open conformation despite environmental changes, whereas those of EM2OH take either the open or folded conformation depending on the polarity or acidity of the solvent This means that stable conformers of EM2 are restricted into a more limited region than those of EM2OH The present study also showed that the aromatic side chains of EM2 have a certain amount of positional freedom, and would therefore occupy similar spatial orientations despite the difference between cis and trans rotamers To investigate the extent of conformational similarity of both rotamers, the spatial orientation of their side chains was compared among possible conformers of cis and trans rotamers, particularly the rS- and n7-type open conformers Consequently, the overall conformational features may be characterized by the spatial orientations of the side chains of the respective residues, and the relative orientation of the aromatic rings of Tyr1 with reference to Phe3 and Phe4 is dependent on the cis and trans EM2 rotamers, as would be surmised from the comparison of Figs and As the presence of (a) a cationic amino group and a phenolic group of Tyr1, (b) the aromatic Phe3 and Phe4 residues, and (c) the C-terminal amidation are necessary for the opioid activity of EM2 [3], the active conformation of EM2 should be taken into consideration on the basis of not only the conformational difference in the backbone chain but also the overall conformation, including the spatial orientation of the respective residues As the rS-type open conformer among the four different backbone folding groups corresponds to the most frequent conformation in the solution despite the difference of cis and trans rotamers (Table 2), it seems important to consider the association of this conformer with bioactive conformation Possible bioactive conformation of EM2 and its comparison with EM2OH X-ray crystal structure analysis is a powerful approach to considering a possible bioactive conformation of an opioid peptide, because it provides well-defined 5090 FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS Y In et al Conformational comparison of endomorphin-2 and its C-terminal free acid Fig Stereoscopic views of conformers A and B observed in the crystal structure of EM2OH, together with hydrogen-bonding water molecules Intermolecular hydrogen bonds are shown by broken lines The displacement ellipsoids are drawn at 70% probability level The atomic numbering of water molecules is also given as W1–W7 molecular conformations at the atomic level As for the l-opioid receptor-specific agonists, two different crystals have been reported, i.e d-TIPP-NH2 (Tyr-dTic-Phe-Phe-NH2) [17] and [Chx2]EM2 (Tyr-Chx-PhePhe-NH2) [18] The similarity between their molecular conformations is shown in Fig Although the spatial orientations of the aromatic rings of their respective residues are somewhat different, these backbone structures take similar folded conformations stabilized by a (Tyr)C ¼ O HN(C-terminal amide or Phe4) hydrogen bond, and are almost the same as the F1-type folded structure of EM2 Therefore, it may be reasonable to consider that (a) the F1-type folded structure is a possible bioactive conformation of EM1 or EM2 and (b) the C-terminal amide NH group plays a role in intramolecular hydrogen bond formation, which is necessary for the folded structure of EM1 or EM2 However, it should be noted that the X-ray conformation of EM2OH showed a similar folded conformation FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS (Fig 8), although its l-opioid receptor agonist activity was completely inhibited by the substitution of the carboxyl group for C-terminal amide group (Table 1) No notable differences were observed for the folded backbone conformation and spatial orientation of aromatic side chains between EM2OH and d-TIPP-NH2, except for the hydrogen-bonding mode between the N- and C-terminal moieties, i.e EM2OH forms a hydrogen bond between the N-terminal amino group and the oxygen atom of the C-terminal carboxyl group, whereas the C-terminal amide NH forms a hydrogen bond with the carbonyl oxygen atom of Tyr1 in the case of d-TIPP-NH2 On the other hand, the conformation of [Chx2]EM2 did not form such a C-terminal NH2-participating hydrogen bond, although it still exhibits the potent l-opioid receptor agonist activity Therefore, the conformational comparison of these three peptides suggests that it may be erroneous to consider a single folded conformation 5091 Conformational comparison of endomorphin-2 and its C-terminal free acid Y In et al Table Torsion angles of cis EM2OH conformers Conformer Tyr1 Pro2 Phe3 Phe4 A B A B A B A B / w x v1 v2 v3 )96.0(4) )92.2(4) )109.3(4) )105.8(4) )136.3(4) )76.1(4) 140.7(4) 134.1(4) )1.5(4) 9.4(4) )50.4(4) )139.6(4) )20.1(4) )32.5(4) 4.7(4) 8.0(4) 177.7(4) 173.7(4) )176.1(4) 167.4(4) 169.8(4) )176.7(4) 36.5(4) 37.8(3) )58.6(4) )56.8(4) )57.5(4) )60.1(4) )85.6(4) )96.4(4) )37.9(4) )40.1(4) )80.0(4) )79.6(4) )73.9(5) )95.8(5) v4 v5 24.0(4) 26.3(3) )0.8(3) )2.1(3) )22.2(3) )22.2(3) Table Intermolecular hydrogen bonds and selected short contacts of cis EM2OH ˚ Length (A) Donor at x,y,z D-H Acceptor A Hydrogen bonds N(1)A O(4¢¢)A N(1)A O(4W) N(1)A O(7W) O(1H)A O(4¢¢)B N(3)A O(2W) N(4)A O(2W) N(1)B O(5W) N(1)B O(6W) N(1)B O(3 W) O(1H)B O(4¢)A N(3)B O(6W) O(1W) O(1¢)A O(1W) O(3 W) O(2W) O(3¢)A O(2W) O(1W) O(3W) O(4¢¢)A O(3W) O(1H)B O(4W) O(5 W) O(4W) O(1H)A O(5W) O(1¢)B O(6W) O(4¢¢)B O(6W) O(3¢)B O(7W) O(4¢)B O(7W) O(4W) Electrostatic short contacts N(1)A O(1W) N(3)A N(2)A O(1H)A O(4W) O(1H)A O(4¢)B N(1)B O(7W) N(3)B N(2)B O(2W) O(4¢¢)A O(3W) O(4¢)A O(4W) O(4¢)B O(4W) N(1)A O(4W) O(1W) O(5W) O(4¢)A O(6W) C(1¢)B 5092 D .A H .A Angle(°) < D-H .A Symmetry operation of A 2.728(5) 2.848(5) 2.754(5) 2.598(5) 2.899(5) 2.936(5) 2.825(5) 2.857(5) 2.778(5) 2.592(5) 2.968(5) 2.773(4) 2.871(4) 2.756(5) 2.822(4) 2.682(5) 2.981(5) 2.796(4) 3.016(4) 2.791(5) 2.765(5) 2.886(5) 2.760(5) 2.831(4) 1.85 1.97 1.91 1.66 2.13 1.88 1.98 2.02 1.97 1.75 2.28 1.76 1.98 1.82 1.94 1.86 2.07 2.04 2.19 1.93 2.01 2.16 1.87 1.90 154 155 154 157 146 174 165 158 148 139 139 167 148 167 173 172 162 138 145 164 156 146 170 164 x,y,z x +1,y,z x +1,y,z 1-x,y +1 ⁄ 2,-z x,y,z x,y,z x,y,z x,y,z x-1,y,z 1-x,y-1 ⁄ 2,1-z x,y,z x,y +1,z x,y +1,z x,y-1,z x,y-1,z x,y,z 1-x,y-1 ⁄ 2,1-z x,y,z 1-x,y +1 ⁄ 2,-z x,y +1,z x,y,z x,y-1,z x,y,z x,y-1,z 3.024(4) 2.783(5) 3.016(4) 3.138(5) 3.021(5) 2.776(5) 3.273(5) 3.122(5) 2.814(4) 2.848(5) 3.221(4) 2.767(5) 3.083(5) x,y,z x,y,z 1-x,y-1 ⁄ 2,-z 1-x,y-1 ⁄ 2,-z x,y,z x,y,z x,y,z x,y-1,z x,y,z x-1,y,z x-1,y,z x-1,y,z x,y,z FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS Y In et al Conformational comparison of endomorphin-2 and its C-terminal free acid Fig Stereoscopic superimpositions of EM2OH (green, molecules A and B), D-TIPPNH2 (red, molecules A and B) and [Chx2]EM2 (black) The backbone chains are represented by thick bonds, and the intramolecular hydrogen bonds are shown by dotted lines stabilized by (Tyr)C ¼ O HN(C-terminal amide) as the bioactive form of EM2 Additionally, the folded conformation may not be bioactive structure of EM2 as it is likely that the most clear-cut conformational difference between EM2 and EM2OH was observed in membrane-mimetic DPC micelles under a physiological condition of pH 5.2, i.e an open conformation for EM2 and a folded conformation for EM2OH On the basis of these results and discussion, we propose that the C-terminal amide group, which is necessary for the l-opioid receptor agonist activity, may play a role in the interaction with the receptor, rather than in forming the bioactive structure; this possibility is also supported by the report on the possible function of the C-terminal amide group in regulating the receptor binding and the agonist ⁄ antagonist property of EM2 [19] If we could correlate the drastic difference between the l-opioid receptor agonist activities of EM2 and EM2OH (Table 1) with their conformational features (Table 2), it would be reasonable to consider the open form, especially the rS-type form, as the most probable opioid-receptor-bound conformation via the C-terminal amide group Docking study of rS-type open conformer of cis and trans EM2 to l-opioid receptor model The representative conformers in the four groups of cis and trans EM2 rotamers were attempted for a docking study of a l-opioid receptor model Some key residues defining the l-opioid receptor binding pocket have already been determined by site-directed mutagenesis studies [20,21], and the importance of Asp147, Tyr148, Glu229, His297, Trp318 and Tyr326 has been indicated for l-opioid receptor agonist activity Therefore, the possible docking site of EM2 was surveyed in such a way that each residue of EM2 interacts with these residues of the receptor model as much as possible Since the mutation of His297 has been reported to FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS result in no detectable binding with l- and d-opioid receptor ligands, we considered that this residue participates in binding with Tyr1 of EM2, because the presence of Tyr1 (its cationic amino and phenolic groups) is necessary for both the l- and d-opioid receptor agonist activities Thus, Tyr1 was set at the bottom of the pocket to form an electrostatic interaction or hydrogen bond between the Tyr1 amino NH3+ or phenolic OH and the His297 imidazole ring On the other hand, Glu229 was considered as a possible residue for interacting with the C-terminal amide of EM2, because it is located at the entrance of the pocket and appears to be feasible for the interaction because of its presence in a flexible loop structure; Asp147 would not be suitable for the interaction with the C-terminal amide, because it is located close to His297 The fitting of the overall conformation of EM2 to the spatial area of the l-opioid receptor pocket showed a high preference for the accommodation of the open conformer of EM2 over the folded conformer Thus, several docking models were constructed for the open conformers and subjected to energy evaluation for reasonable configuration and translation exploration using the Docking module of insight discover Consequently, it was suggested that the pocket shape and size of the model receptor is most suitable for accommodating the rS-type open conformer of trans EM2, as shown in Fig 9, where hydrogen bond formations are possible between (Tyr1) NH O(Asp147) (Tyr1)OH imidazole N(His297) (Tyr1)OH O(Ala240) (Tyr1)O HO(Tyr148) (Tyr1)O HO(Thr218) and (C-amide)NH O (Glu229) pairs, and the Trp293 and Trp318 indole rings form stacking interactions with Tyr1, Phe3 and Phe4 aromatic rings, contributing to the binding stabilization of EM2 to the pocket It appears important to note that this binding model is stereospecific, and the mirror-imaged conformer of trans EM2 or the open conformer of cis EM2 leads to a labile binding due to the breakage of these interactions 5093 Conformational comparison of endomorphin-2 and its C-terminal free acid Y In et al Fig Stereoscopic view of possible docking of rS-type open conformer of EM2 (ball and stick model) on the agonist binding site of the l-opioid receptor structural model (green ribbon model) The functional residues of the receptor for the interaction are shown by a ball and stick model The hydrogen bonds or electrostatic interactions are represented by dotted lines In conclusion, the present study has demonstrated that EM2 prefers to form the open conformation and the substitution of a free acid for the C-terminal amide increases the population ratio of the folded conformers in solution, despite its electronic state (zwitterionic or monocationic form), although the open and folded conformational features, except for population ratios, are not significantly different between EM2 and EM2OH The conformation–activity relationship of EM2 and EM2OH suggests that the F1-type folded conformation stabilized by a (C-terminal amide) HN O ¼ C(Tyr) hydrogen bond is not the bioactive structure of EM Alternatively, the rS-type open conformation of trans EM was suggested to be important for the interaction with the receptor via the C-terminal amide group on the basis of the docking study of EM2 to a l-opioid receptor model These results would provide a structural–conformational reason why the C-terminal amidation of EM is necessary for its l-opioid receptor agonist activity Experimental procedures Peptides EM1, EM2, EM1OH and EM2OH were synthesized and purified according to a previous report [6] and checked for homogeneity by analytical HPLC and amino acid analysis, and were judged to be > 95% pure ously [22] For the binding assay, synaptosomal brain membrane P2 preparations from Sprague–Dawley rats were used as sources of l- and d-opioid receptors after the removal of endogenous opioids The competitive displacement assay used 3.5 nm [3H]Tyr-d-Ala-Gly-MePhe-Gly-ol and 5.57 nm [3H][d-penicillamine 2-d-penicillamine 5]enkephalin for the l- and d-sites, respectively, and the affinity constants (Ki) were determined using a conventional procedure On the other hand, for the in vitro guinea pig ileum bioassay, the myenteric plexus-longitudinal muscle was obtained from a male Hartley strain guinea pig ileum and the tissue was mounted in a 10-mL bath that contained aerated (95% O2, 5% CO2, v ⁄ v) Krebs–Henseleit solution at 35 °C The tissue was stimulated transmurally between platinum wire electrodes using pulses of 0.5 ms duration with a frequency of 0.1 Hz at supramaximal voltage Longitudinal contractions were recorded via an isometric transducer For the mouse vas deferens bioassay, the vas deferentia of a male ddY strain mouse were prepared The vas deferentia were mounted in a 10-mL bath containing aerated, modified Mg2+-free Krebs solution containing 0.1 mm ascorbic acid and 0.027 mm EDTA4Na at 35 °C The tissue was stimulated transmurally with trains of rectilinear pulses of ms Stimulation trains were given at 20-s intervals and consisted of seven stimuli of 1-ms duration with 10-ms intervals In both bioassays, dose–response curves were constructed, and IC50 values (concentration causing a 50% reduction in the number of the electrically induced twitches) were calculated NMR measurements Opioid activity measurements The binding assays and in vitro bioassays of EM1, EM2, EM1OH and EM2OH were performed as described previ- 5094 H-NMR spectra were recorded on a Varian unity INOVA500 spectrometer with a variable temperature-control unit EM2 or EM2OH was dissolved in 0.7 mL of TFE FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS Y In et al Conformational comparison of endomorphin-2 and its C-terminal free acid solvent, H2O ⁄ D2O (9 : 1), or DPC micelles, where the 40-fold excess molar ratio of perdeuterated DPC to EM2 or EM2OH is dissolved in H2O ⁄ D2O (9 : 1) The concentrations used for NMR measurements were 10 mm for TFE, 16 mm for H2O ⁄ D2O, and 13 mm for DPC micelles The pH of the peptide solutions directly dissolved in water and DPC micelles were 2.6 and 3.4, respectively NMR measurements were also performed for the water and DPC micelles solutions adjusted to pH 5.2 by adding 10 mm NaOH, to confirm the conformation of EM2 or EM2OH in the neutral or zwitterionic form, respectively Chemical shifts were measured as downfield shifts (in p.p.m) from internal TSP-d4 (2,2,3,3-tetradeuterio-3-(trimethylsilyl)propionic acid sodium salt) The solutions were degassed and sealed under vacuum All NMR measurements were performed at 25 °C The temperature dependence of the chemical shift of each NH proton was measured in the range of 20–60 °C (10 °C intervals) The NMR measurements were performed by the same procedure used in dimethyl sulfoxide [6] Two-dimensional COSY, TOCSY and ROESY were acquired in the phasesensitive mode using standard pulse programs available in the Varian software library Continuous low-power irradiation was performed during the relaxation delay and the mixing time to suppress the peak due to water Spectra were zero-filled to achieve a digital resolution of 0.4 Hz per point The TOCSY and ROESY spectra were recorded with mixing times of 80 ms and 300 ms, respectively ROE intensities were classified into three groups (strong, medium, and weak) to estimate proton-proton distance A possible torsion angle was estimated from the vicinal coupling constants using the equations 3JHNCaH ¼ 9.8cos2h–1.1cosh +0.4sin2h, where / ¼ |h–60|° for the / torsion angle around the CÂi-1NiCaiCÂi bond sequence [23] and JHCaCbH ẳ 11.0cos2h)1.4cosh +1.6sin2h for the h angle around the H–Cai–Cbi–H bond [24] Computational molecular modelling calculation The constructions of 3D molecular conformations, which satisfied the ROE constraints within allowable range, were performed using the dynamical simulated annealing method [25,26] with insight ii ⁄ discover software [27] according to a previous paper [6] By the steepest descent and successive conjugate gradient method, conformational energy was minimized The system was simulated for 50 ps at 1000 K by solving Newton’s equation of motion The global minimum of the target function consisting of force constants for the covalent bond (Fcovalent), repulsive van der Waals’ contact (Frepul), chirality (Fchiral), torsion (Ftor), and interproton distance (FROE) was searched by initially and substantially increasing the force constants until they regained their full values; the force constant for chirality (Fchiral) was kept constant during simulated annealing calculation to avoid the swapping the chiralities at the high FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS temperature simulation After this process, temperature was decreased stepwise until 300 K During this process, the van der Waals’ repulsion term was set to be predominant After that, the structure was again energy-minimized to refine the conformer As input data for the FROE constraints, the proton– proton pairs were classified into three distance groups ˚ according to ROE intensity: strong (1.6–2.6 A), medium ˚ ) and weak (1.6–5.0 A) Since the aromatic pro˚ (1.6–3.5 A tons of Phe3 and Phe4 were not separately assigned, their ROEs were treated for three cases, i.e either contribution of Phe3 or Phe4, or the simultaneous contribution of both residues The (< r6 >))1 ⁄ distance averaging method was used for the equivalent protons For distance constraints involving aromatic ring protons of Phe3 and Phe4, which were not stereospecifically assigned, the pseudoatom treatment was used Other potential functions were all calculated according to the protocol from insight ii ⁄ discover 2000 software The restraints for the / and h torsion angles were not included in the calculations; they were used as indicators to estimate the reliability of the constructed 3D structures X-ray crystal analysis of EM2OH Single crystals of EM2OH were grown from an aqueous solution (pH 5.2) at room temperature in the form of transparent and colourless needles A crystal 0.6 · 0.1 · 0.01 mm3 was mounted on a nylon loop with 30% glycerol of mother liquor and then flash-frozen under a nitrogen stream (120 K) Data collection was performed on a CCD diffractometer (Bruker AXS SMART APEX) The crystal data are as follows: C32H36N4O6ặ3.5H2O, Mr ẳ 635.70, ˚ monoclinic, P21, a ¼ 19.687(2) A, b ¼ 6.5058(7) A, c ¼ ˚ , b ¼ 101.370(2)°, V ¼ 3248.3(6) A3, Z ¼ 4, ˚ 25.869(3) A F000 ¼ 1356, l(Mo Ca) ¼ 0.096 mm)1, number of observed reflections ¼ 20743, Rint ¼ 0.0453, number of reflections used for refinement ¼ 9580, number of parameters ¼ 819, final R ¼ 0.0699 and wR ¼ 0.1391 (d ⁄ r)max ¼ 0.001, ˚ ˚ Dqmax ¼ 0.367 eA)3, and Dqmin ¼ )0.342 eA)3 The crystal structure was solved by direct methods using the shelxs-97 program [28] The atomic scattering factors were taken from International Tables for X-ray Crystallography [29] The positional parameters of non-H atoms were refined by a full-matrix least-squares method with anisotropic thermal parameters using the shelxs-97 program [30] The positions of H atoms of amino and hydroxyl groups of EM2OH and water molecules were determined from a difference Fourier map, while those of the other H atoms were calculated on the basis of their stereochemical requirement They were treated as riding with fixed isotropic displacement parameters (Uiso ¼ 1.2 Ueq for the associated C or N atoms, or Uiso ¼ 1.5 Ueq for O atoms) and were not included as variables for the refinements The final crystallographic information file (cif) data involving 5095 Conformational comparison of endomorphin-2 and its C-terminal free acid atomic coordinates, anisotropic temperature factors, bond lengths, bond angles, torsion angles of non-H atoms, and atomic coordinates of H atoms have been deposited in the Cambridge Crystallographic Data Center (CCDC 271791), Cambridge University Chemical Laboratory, Cambridge, UK Model building of l-opioid receptor As an experimentally determined 3D structure of a l-opioid receptor is not yet available, its 3D model was generated using modeler (DS Modeling, Accelrys Software Inc., San Diego, CA) according to the protocol of comparative modelling [31], where the X-ray crystal structure of rhodopsin (PDB code: 1f88) was selected as the template structure from the Protein Data Bank using a Gapped Blast of Protein Similarity Search module Model building was followed by energy minimization using CHARMm (DS Modeling), choosing CHARMm22 as a force field On the other hand, an agonist peptide-incorporated structural model of the l-opioid receptor has already been constructed by Mosberg et al [20] (model title: OPRM_RAT_AD_JOM6) and is available from the laboratory home page Thus, this model was compared with our constructed model, and no notable discrepancy was observed The initial docking of EM2 on the pocket of the l-opioid receptor model was visually performed on a graphics computer while keeping the conformation, and then the possible docking mode was refined using the Docking module of insight discover, where the energy evaluation was performed for the reasonable configuration and translation exploration 10 11 Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Sciences, and Technology of Japan (Y.I.) 12 References 13 Eipper BA & Mains RE (1988) Peptide alpha-amidation Annu Rev Physiol 50, 333–344 Yang YR, Chiu TH & Chen CL (1999) Structure-activity relationships of 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models: The structural basis of mu vs delta selectivity J Peptide Res 60, 329–335 Mansour A, Taylor LP, Fine JL, Thompson RC, Hoversten MT, Mosberg HI, Watson SJ & Akil H (1997) Key residues defining the l–opioid receptor binding pocket: a site-directed mutagenesis study J Neurochem 86, 344–353 Fujita Y, Tsuda Y, Li T, Motoyama T, Takahashi M, Shimizu Y, Yokoi T, Sasaki Y, Ambo A, Kita A, Jinsmaa Y, Bryant SD, Lazarus LH & Okada Y (2004) Development of potent bifunctional endomorphin-2 analogues with mixed l- ⁄ d-opioid agonist and d-opioid antagonist properties J Med Chem 47, 3591– 3599 Bystrov VF (1976) Spin-spin coupling and the conformational states of peptide systems Prog Nucl Magn Reson Spectrosc 10, 41–81 Kopple KD, Wiley GR & Tauke R (1973) Dihedral angle-vicinal proton coupling constant correlation for the a–b bond of amino acid residues Biopolymers 12, 627–636 Clore GM, Nigles M, Sukumaran DK, Bruenger AT, Karplus M & Gronenborn AM (1986) The three- FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS 26 27 28 29 30 31 dimensional structure of a1-purothionin in solution: combined use of nuclear magnetic resonance, distance geometry and restrained molecular dynamics EMBO J 5, 2729–2735 Nilges M, Clore GM & Gronenborn AM (1988) Determination of three-dimensional structures of proteins from interproton distance data by hybrid distance geometry-dynamical simulated annealing calculations FEBS Lett 229, 317–324 INSIGHT ⁄ DISCOVER Accelrys Software Inc, San Diego, CA Sheldrick GM (1997) SHELXS97 Program for the Refinement of Crystal Structures University of Gottingen, Germany Edited by Theo Hahn (1992) International Tables for X-Ray Crystallography Vol C Kluwer Academic Publishers, Dordrecht Sheldrick GM (1997) SHELXL97 Program for the Solution of Crystal Structure University of Gottingen, Germany Fiser A & Sali A (2003) Modeller: generation and refinement of homology-based protein structure models Methods Enzymol 374, 461–491 Supplementary material The following material is available for this article online: Fig S1 Temperature dependence of NH protons in TFE, water (pHs 2.7 and 5.2), and DPC micelles (pHs 3.5 and 5.2) Fig S2 Stereoscopic molecular packing figures of EM2OH in the crystal structure, viewed from b-axis Table S1 Chemical shifts and coupling constants of respective protons in TFE, water (pHs 2.7 and 5.2), and DPC micelles (pHs 3.5 and 5.2) Table S2 Interresidual ROE connectivities along the peptide backbones in TFE, water (pHs 2.7 and 5.2), and DPC micelles (pHs 3.5 and 5.2) Table S3 Bond lengths, bond angles, and torsion angles of EM2OH molecules in the crystal structure 5097 ... 2.8 31( 4) 1. 85 1. 97 1. 91 1.66 2 .13 1. 88 1. 98 2.02 1. 97 1. 75 2.28 1. 76 1. 98 1. 82 1. 94 1. 86 2.07 2.04 2 .19 1. 93 2. 01 2 .16 1. 87 1. 90 15 4 15 5 15 4 15 7 14 6 17 4 16 5 15 8 14 8 13 9 13 9 16 7 14 8 16 7 17 3 17 2 16 2... 16 2 13 8 14 5 16 4 15 6 14 6 17 0 16 4 x,y,z x +1, y,z x +1, y,z 1- x,y +1 ⁄ 2,-z x,y,z x,y,z x,y,z x,y,z x -1, y,z 1- x,y -1 ⁄ 2 ,1- z x,y,z x,y +1, z x,y +1, z x,y -1, z x,y -1, z x,y,z 1- x,y -1 ⁄ 2 ,1- z x,y,z 1- x,y... al 17 18 19 20 21 22 23 24 25 Conformational comparison of endomorphin- 2 and its C-terminal free acid endomorphin- 1 using NMR spectroscopy and molecular modeling FEBS Lett 439, 13 –20 Flippen-Anderson