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
1 Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka, Japan 2 Department of Biochemistry, Tohoku Pharmaceutical University, Sendai, Japan 3 Medicinal Chemistry Group, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA 4 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 8 August 2005, accepted 16 August 2005)
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 meas- urement and molecular modelling calculation. Both peptides were in equi- librium between the cis and trans rotamers around the Tyr–Pro w bond with population ratios of 1 : 1 to 1 : 2 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 2 : 1. Fifty possible 3D conformers were generated for each peptide, taking dif- ferent 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 pro- ton-proton distance constraints derived from the ROE cross-peak intensi- ties. These conformers were then roughly classified into four groups of two open [reverse S (rS)- and numerical 7 (n7)-type] and two folded (F1- and F2-type) conformers according to the conformational pattern of the back- bone structure. Most EM2 conformers in neutral (in TFE) and monocati- onic (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 F1- type structure similar to that stabilized by an intramolecular hydrogen +...COO–(Phe4), observed in its crystal structure. These bond of (Tyr1)NH3 results show that the substitution of a carboxyl group for the C-terminal
doi:10.1111/j.1742-4658.2005.04919.x
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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.
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 con- formational features of EM2 and EM2OH and a possible model for bind- ing to the l-opioid receptor, constructed from the template structure of rhodopsin.
Many bioactive peptides are a-amidated at the C ter- minus. 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 under- stood 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], assu- ming that C-terminal amidation is significantly asso- ciated with the bioactive conformation of a peptide or its interaction with a receptor.
free acid [8]. Furthermore,
agonist TIPP-NH2
N-Terminal amidated endomorphin-1 (EM1, Tyr- Pro-Trp-Phe-NH2) and endomorphin-2 (EM2, Tyr- Pro-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 (Tyr- Pro-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 the d-opioid receptor selectivity of the l-opioid receptor- (Tyr-Tic-Phe-Phe-NH2, specific where Tic ¼ tetrahydro-3-isoquinoline carboxylic acid) was reported to be increased significantly if the C-ter- minal amide was replaced by a free acid [9]. Therefore, differentiation between the l- and d-opioid receptor- selective peptides results from the C-terminal region.
of a cationic amino group and a phenolic group in position 1, a spacing amino acid in position 2, lipophi- lic and aromatic residues in positions 3 and 4, and C-terminal amidation [3]. Using this concept, a com- parative 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 deter- mined 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 confor- mation proposed for EM2. This result appears to be important, because it means that C-terminal amida- tion, which shifts the N-terminal amino group to a neutral state, participates in forming a defined bio- active conformation. To confirm whether this phenom- enon 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 features of EM2 and the intrinsic conformational EM2OH, which is important when considering the substrate specificity of l-opioid receptors and the structural role of C-terminal amidation.
Y. In et al. Conformational comparison of endomorphin-2 and its C-terminal free acid
Results
Opioid activity
The binding affinities of EM1, EM2, EM1OH and EM2OH for l- and d-opioid receptors and the
On the other hand, the biological function of natur- ally 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
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Y. In et al. Conformational comparison of endomorphin-2 and its C-terminal free acid
Table 1. 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)
[12]
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 obser- vations 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 2.
TIPP-NH2
results
that
these compounds as pharmacological activities of l- and d-opioid receptor agonists are given in Table 1. Although the bioassays of these peptides by Al-Khrasani et al. 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 dras- tic loss of binding affinity and agonist activity for the l-opioid receptor; a similar decrease of activity has been observed for morphiceptin (Tyr-Pro-Phe- free acid [8]. The Ki Pro-NH2) and its C-terminal 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 the l-opioid receptor-specific receptor selectivity of was (Tyr-Tic-Phe-Phe-NH2) agonist increased significantly if the C-terminal amide was replaced by a free acid. It is obvious from the pre- sent the differentiation between the l- and d-opioid receptor selectivities of EM2 is rela- ted to C-terminal amidation.
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 carb- oxyl group (anionic or neutral) of EM2OH. There- fore, 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).
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 2.
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.
1H-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 diffi- the cult
these aromatic protons. Because of
for
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 tem- perature 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
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0.36 200 0.69 200 EM1 EM1OH EM2 EM2OH 1500 1800 9200 3950 10.1 ± 1.2 4032 ± 4330 5.79 ± 0.4 > 104 36.3 ± 5.2 > 104 344 ± 93 > 104
Y. In et al. Conformational comparison of endomorphin-2 and its C-terminal free acid
Table 2. 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.
DPC H2O
pH 5.2 pH 2.7 pH 3.5 pH 5.2 Solvent electronic form DMSO TEF
EM2 trans Neutral trans ⁄ cis ¼ 3 : 2 – – trans – – trans ⁄ cis ¼ 2 : 1 Open (rS ¼ 30)
Monocation – trans ⁄ cis ¼ 3 : 2 Open (rS ¼ 18, n7 ¼ 4) fold (F2 ¼ 8) – Open (rS ¼ 18, n7 ¼ 12) Open (rS ¼ 17, n7 ¼ 4) fold (F1 ¼ 1, F2 ¼ 8) cis Neutral – – – – Open (rS ¼ 30)
Open (rS ¼ 13, n7 ¼ 7) fold (F2 ¼ 10) Monocation – – – –
EM2OH trans Zwitter trans ⁄ cis ¼ 2 : 1 Fold (F1 ¼ 30)
trans ⁄ cis ¼ 1 : 1 Open (rS ¼ 21, n7 ¼ 2) fold (F2 ¼ 7) trans ⁄ cis ¼ 1 : 1 Open (n7 ¼ 11) fold (F1 ¼ 14, F2 ¼ 5) trans Open (rS ¼ 12, n7 ¼ 9) fold (F1 ¼ 1, F2 ¼ 8) trans ⁄ cis ¼ 2 : 1 Open (rS ¼ 6, n7 ¼ 12) fold (F1 ¼ 7, F2 ¼ 5) – – – Monocation – –
– cis Zwitter Fold (F1 ¼ 30) 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
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.ÆK)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.ÆK)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.ÆK)1), suggesting the participation of this group in any specific interactions (see later discussion).
(pH 2.7), water
To estimate proton–proton distance, ROESY spec- tra were measured according to the short-, medium- and long-range ROE connectivities along the peptide backbone. Some selected inter-residual ROE connecti- vities, which show the characteristic differences between the EM2 and EM2OH, are listed in Table 4. As the long-range ROEs, which have a strong influ- ence on determining the overall molecular confor- mation, were very few, the peptides would be an ensemble of many different conformers. However,
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 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(cid:1). According to solvent two types of electronic state were type and pH,
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Open (rS ¼ 5, n7 ¼ 7) fold (F1 ¼ 18) Monocation – – – – – Open (rS ¼ 7, n7 ¼ 20) (F2 ¼ 2) trans ⁄ cis ¼ 3 : 2 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)
fol-
(F1-type) and nonhydrogen-bonded (F2-type) dings. The results are given in Table 2.
Y. In et al. Conformational comparison of endomorphin-2 and its C-terminal free acid
Conformational characteristics of EM2
Table 3. Temperature coefficients (Dd ⁄ DT, p.p.b.ÆK)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 (–).
DPC H2O
pH 2.7 pH 5.2 pH 3.5 pH 5.2 Residue TFE
EM2 trans
Phe3NH Phe4NH C-term.NH2 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 _ – cis
Phe3NH Phe4NH C-term.NH2 4.58 4.20 3.01 5.11 7.70 7.62 – – 7.70 7.62 – –
EM2OH trans
Phe3NH Phe4NH 3.27 5.68 9.49 6.35 8.63 3.00 7.13 6.56 7.67 4.46 cis
As shown in Table 2, EM2 has a trans ⁄ cis rotamer ratio of about 3 : 2 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 2 : 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 solu- tion 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 condi- tions. As EM1 has a trans ⁄ cis equilibrium of ratio 74 : 26 in SDS micelles and the cis rotamer is predom- inant 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
for EM2 in neutral
twisting at
in which the target
considered: form (N-terminal NH2 and C-terminal NH2) in TFE and in monocati- + and C-terminal NH2) onic form (N-terminal NH3 in water and DPC micelles; for EM2OH in zwitteri- + and C-terminal COO–) onic form (N-terminal NH3 in TFE, water and DPC micelles and in monocation- + and C-terminal COOH) ic form (N-terminal NH3 in water (pH 2.7) (see Table 2). Starting with 50 dif- ferent conformation sets with random arrays of atoms, energy-minimization trials were performed to eliminate any possible source of initial bias in the function was folding pathway, minimized by changing /, w, x and v torsion angles. Although neither of the peptides produced well- refined conformers that agreed perfectly with all con- straints 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 con- stants) within ± 30(cid:1). On the basis of their backbone conformations, the respective conformers were classi- fied into four groups. The open conformers were divided into two groups of ‘numerical 7 (n7)’-like and ‘reverse S (rS)’-like curves; and the fold con- formers were divided into two groups according to the interaction pattern between the C- and N-ter- their hydrogen-bonded minal polar atoms,
that
is,
As the pK1 of Tyr is 2.2, most EM2 conformers are thought to overwhelmingly take the monocationic elec- tronic 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 the Pro2-Phe3 moiety. Many structure, ROEs between neighbouring residues and minor ROEs between residues separated by more than one residue resulted in the absence of a well-defined overall struc- ture, and the lack of direct ROEs among the aromatic protons of Tyr1, Phe3 and Phe4 leads to the fluctu- ation of these rings. The superimposed backbone struc- tures of 30 energetically stable conformers in the respective solutions are shown in Fig. 1, and the most stable conformers that belong to the respective con- formational groups are shown in Fig. 2. As shown in Fig. 1 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 val- ues suggest the other many conformers. Table 2 also shows that the flexibility of the overall conformation increases in the various solutions in the order of di- methylsulfoxide < TFE ¼ water < DPC micelles. The
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Phe3NH Phe4NH 3.93 6.10 7.81 7.34 7.18 4.88 6.47 2.01
Y. In et al. Conformational comparison of endomorphin-2 and its C-terminal free acid
Table 4. 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 Proton j Intensity Proton i Proton j Intensity
trans EM2OH Phe3 NH Weak TFE trans EM2 Tyr1 b2
Pro2 d1 C-NH2 C-NH2 Weak Medium Medium cis EM2OH cis EM2 Tyr1 b2 Phe4 a Phe4 a
Phe3 NH Pro2 d1 Pro2 a Phe4 NH Weak Weak Weak Weak Tyr1 a Tyr1 2,6H Tyr1 3,5H Phe3 b2
H2O (pH 2.7) trans EM2 trans EM2OH Tyr1 2,6H Phe3 2,6H Pro2 d2 Phe4 NH Weak Medium
cis EM2 cis EM2OH
Pro2 d2 Phe4 a C-NH1 C-NH2 Pro2 a Pro2 a Phe3 NH Weak Weak Medium Weak Medium Strong Weak Tyr1 b1 Phe3 3,5H Phe4 a Phe4 a Tyr1 b1 Tyr1 b2 Pro2 c2
Phe3 2,6H Pro2 d1 Phe4 NH Phe4 NH Phe4 NH Medium Weak Medium Strong Medium Tyr1 b2 Tyr1 2,6H Pro2 a Phe3 a Phe3 b2
H2O (pH 5.2) trans EM2 trans EM2OH
Tyr1 3,5H Pro2 b1 Phe3 NH Pro2 c Phe4 NH Phe4 NH Medium Weak Weak
cis EM2 cis EM2OH Pro2 d2 Phe4 a Phe4 NH C-NH1 C-NH2 Phe3 NH Phe3 NH Weak Weak Weak Medium Weak Weak Weak Tyr1 b1 Phe3 3,5H Phe3 b1 Phe4 a Phe4 a Pro2 b2 Pro2 c2
Pro2 a Phe4 NH Phe4 NH Phe4 NH Phe4 NH Medium Weak Weak Medium Medium Tyr1 3,5H Pro2 b1 Phe3 NH Phe3 a Phe3 b2
DPC (pH 3.5) trans EM2 trans EM2OH
Tyr1 2,6H Pro2 b1 Phe3 b2 Phe3 2,6H Phe3 2,6H Phe3 3,5H Phe3 3,5H Phe3 a Phe3 2,6H Phe4 2,6H Phe4 2,6H Phe4 3,5H Phe4 a Phe4 2,6H Weak Weak Weak Medium Medium Medium Medium Phe4 2,6H Phe4 3,5H Phe4 2,6H Phe4 3,5H Phe4 b2 Phe4 2,6H Phe4 NH Weak Weak Weak Weak Weak Medium Weak Pro2 b1 Pro2 b1 Pro2 c1 Pro2 c1 Phe3 a Phe3 a Phe3 b2
DPC (pH 5.2) trans EM2 trans EM2OH
Phe3 NH Phe3 2,6H Phe4 2,6H Phe4 NH Weak Weak Weak Weak Pro2 b1 Pro2 c1 Phe3 b1 Phe3 b2
Tyr1 2,6H Phe3 2,6H Phe3 2,6H Phe3 2,6H Phe3 3,5H Phe3 3,5H Phe3 a Phe4 a Phe4 3,5H Phe4 2,6H Phe4 a Phe4 2,6H Weak Weak Medium Medium Medium Medium cis EM2OH
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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 Tyr1 a Tyr1 2,6H Tyr1 2,6H Tyr1 3,5H Pro2 a Phe3 NH Phe3 a Phe3 b1 Phe3 b2
Cis EM2
Y. In et al. Conformational comparison of endomorphin-2 and its C-terminal free acid
A
B
is noteworthy that
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 conforma- tional groups are shown in Fig. 4. The cis EM2 con- formers 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 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 sug- gest 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 well- defined rS-type open conformation despite the differ- ence of cis and trans rotamers.
C
In conclusion, this study showed that the solution conformation of EM2 could be grouped into four con- formers, 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.
Conformational characteristics of EM2OH
circumstance, as
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 1 : 1 to 2 : 1 in TFE, water and neutral DPC micelles (pH 5.2), and characteristically EM2OH in DPC micelles of pH 3.5
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 compared to DMSO, TFE or water.
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Fig. 1. 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 con- formations are overlaid so as to superimpose their Tyr-Pro back- bone chains.
Y. In et al. Conformational comparison of endomorphin-2 and its C-terminal free acid
existed almost completely as a trans rotamer, similar to the case of trans EM2.
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.
Trans EM2OH
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 struc- ture, and this is due to the disappearance of the elec- trostatic interactions between the cationic N-terminal and anionic C-terminal groups.
Cis EM2OH
The superimposed backbone structures of 30 energetic- ally 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. 2. 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
The superimposed backbone structures of 30 energetic- ally 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. 4. In DPC micelles, the cis rotamer of EM2OH existed only in neutral (pH 5.2) with a trans ⁄ cis ratio of 2 : 1. As is obvious from Table 2, most conformers of cis EM2OH in all solu- tions preferred to take the F1-type folded conforma- tion through the NH...O hydrogen bond between the N- and C-terminal ends, although the equilibrium with
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Fig. 2. Stereoscopic views of most stable conformers of trans EM2 belonging to respective conformational groups. (A) rS-type open con- former in TFE, (B) n7-type open conformer in water, (C) F1-type and (D) F2-type folded conformers in DPC micelles.
Y. In et al. Conformational comparison of endomorphin-2 and its C-terminal free acid
A
B
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 trans rS-type open form, similarly to the case of EM2OH.
Crystal structure of EM2OH
+... mainly stabilized by an intramolecular (Tyr1)NH3 –OOC(Phe4) hydrogen bond, in addition to the electro- static interaction of the (Pro2)N...NH(Phe3) atomic pair. A water molecule (O2W) is bifurcately hydrogen- bonded to the two NH protons of Phe3 and Phe4 resi- dues, playing an auxiliary role in stabilizing this F1 conformation. On the other hand, such an intramole- cular 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 +(Tyr1), NH(Phe3) water molecule (O6W) with NH3 and –OOC(Phe4), in addition to the indirect interac- tion between both terminal polar groups via a water +(Tyr1) electrostatic molecule (O7W): a O7W...NH3 interaction and a O7W...OOC– (Phe4) hydrogen bond.
The EM2OH crystal consists of two independent con- formers (conformers A and B) and seven water mole- cules per asymmetric unit. These conformers are torsion shown in Fig. 7. Selected conformational angles, hydrogen bonds and electrostatic short contacts are given in Tables 5 and 6. 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
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Fig. 3. Stereoscopic superimpositions of backbone structures of 30 energetically sta- ble conformers of cis EM2 in (A) TFE and (B) water (pH 2.7 and 5.2). The conforma- tions are overlaid so as to superimpose their Tyr-Pro backbone chains.
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-ter- minal amide group increased the population of folded conformer in the molecular conformation, which lar- gely 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 monocation- ic 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 char- acteristic 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.
Y. In et al. Conformational comparison of endomorphin-2 and its C-terminal free acid
Fig. 4. 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
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 con- formation similarly to its trans rotamer, except the ori- entation 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) overwhelm- ingly 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.
NMR analyses indicated that both EM2 and EM2OH are in equilibrium between open and folded conform- ers with trans ⁄ cis population ratios of 1 : 1 to 2 : 1 in dimethyl sulfoxide, TFE and water, although the fre- quency of taking the cis rotamer of EM2OH is higher than that of EM2. In contrast, EM2 takes only the
The present study demonstrates that conformers of to take the open conformation. The EM2 prefer rS-type open conformer of trans EM2, such as that in Fig. 2A, exists as the major conformer in all solutions,
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Y. In et al. Conformational comparison of endomorphin-2 and its C-terminal free acid
A
B
C
D
E
F
In the
conformational preference.
+ and C-terminal bond) between the N-terminal NH3 COO– groups acts as a driving force for folding the molecular conformation. In other words, the C-ter- minal amide group plays a role in preventing the for- mation of such a folded conformation.
Single crystals of EM2OH were successfully obtained from neutral water adjusted to pH 5.2 by NaOH addi- tion, whereas the crystals were not obtained from water (pH 2.3). The crystal analysis showed two inde- pendent, but similar F1-type folded conformers of zwitterionic cis EM2OH, which also existed in dimeth- yl sulfoxide, TFE, water, or DPC micelles, indicating that this F1-type folded conformer is one of the most stable conformers of EM2OH.
and the cis rotamer could also be classified with the same 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
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Fig. 5. 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.
Y. In et al. Conformational comparison of endomorphin-2 and its C-terminal free acid
(B) water (C) water (pH 5.2), (pH 2.7),
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 con- changes, whereas environmental formation despite those of EM2OH take either the open or folded confor- mation depending on the polarity or acidity of the sol- vent. 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 aro- matic 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 con- formational similarity of both rotamers, the spatial ori- entation of their side chains was compared among possible conformers of cis and trans rotamers, partic- ularly the rS- and n7-type open conformers. Conse- quently, the overall conformational features may be characterized by the spatial orientations of the side chains of the respective residues, and the relative orien- tation 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 2 and 4. 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 amida- tion are necessary for the opioid activity of EM2 [3], the active conformation of EM2 should be taken into con- sideration 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 cor- responds to the most frequent conformation in the solu- tion 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 it provides well-defined opioid peptide, because
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Fig. 6. Stereoscopic superimpositions of backbone structures of 30 energetically stable conformers of zwitterionic cis EM2OH in (A) TFE, (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 back- bone chains.
Y. In et al. Conformational comparison of endomorphin-2 and its C-terminal free acid
hydrogen
(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 aro- matic 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 bond, although it still exhibits the potent l-opioid receptor agonist activity. Therefore, the conformational com- parison of these three peptides suggests that it may be erroneous to consider a single folded conformation
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-d- Tic-Phe-Phe-NH2) [17] and [Chx2]EM2 (Tyr-Chx-Phe- Phe-NH2) [18]. The similarity between their molecular conformations is shown in Fig. 8. Although the spatial orientations of the aromatic rings of their respective residues are somewhat different, these backbone struc- tures take similar folded conformations stabilized by a (Tyr)C ¼ O...HN(C-terminal amide or Phe4) hydro- gen bond, and are almost the same as the F1-type folded structure of EM2. Therefore, it may be reason- able 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 conforma- tion of EM2OH showed a similar folded conformation
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Fig. 7. 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 dis- placement ellipsoids are drawn at 70% pro- bability level. The atomic numbering of water molecules is also given as W1–W7.
Y. In et al. Conformational comparison of endomorphin-2 and its C-terminal free acid
Table 5. Torsion angles of cis EM2OH conformers.
Conformer / w x v1 v2 v3 v4 v5
Tyr1
Pro2 24.0(4) 26.3(3) )0.8(3) )2.1(3) )22.2(3) )22.2(3) Phe3 4.7(4) 8.0(4) 177.7(4) 173.7(4) )176.1(4) 167.4(4) Phe4 A B A B A B A B 140.7(4) 134.1(4) )1.5(4) 9.4(4) )50.4(4) )139.6(4) )20.1(4) )32.5(4) )96.0(4) )92.2(4) )109.3(4) )105.8(4) )136.3(4) )76.1(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)
Table 6. Intermolecular hydrogen bonds and selected short contacts of cis EM2OH.
Length (A˚ )
Donor at x,y,z D-H Acceptor A D. . .A H. . .A Symmetry operation of A Angle((cid:1)) < D-H. . .A
Hydrogen bonds
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 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) O(4¢¢)A O(4W) O(7W) O(4¢¢)B O(2W) O(2W) O(5W) O(6W) O(3 W) O(4¢)A O(6W) O(1¢)A O(3 W) O(3¢)A O(1W) O(4¢¢)A O(1H)B O(5 W) O(1H)A O(1¢)B O(4¢¢)B O(3¢)B O(4¢)B O(4W) N(1)A N(1)A N(1)A O(1H)A N(3)A N(4)A N(1)B N(1)B N(1)B O(1H)B N(3)B O(1W) O(1W) O(2W) O(2W) O(3W) O(3W) O(4W) O(4W) O(5W) O(6W) O(6W) O(7W) O(7W) 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 Electrostatic short contacts
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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) N(1)A N(3)A O(1H)A O(1H)A N(1)B N(3)B O(2W) O(3W) O(4W) O(4W) O(4W) O(5W) O(6W) 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 O(1W) N(2)A O(4W) O(4¢)B O(7W) N(2)B O(4¢¢)A O(4¢)A O(4¢)B N(1)A O(1W) O(4¢)A C(1¢)B
Y. In et al. Conformational comparison of endomorphin-2 and its C-terminal free acid
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 pro- pose that the C-terminal amide group, which is neces- sary 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 func- tion 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 differ- ence 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.
is most
Docking study of rS-type open conformer of cis and trans EM2 to l-opioid receptor model
(Tyr1)OH...O(Ala240)
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 indica- ted 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
result in no detectable binding with l- and d-opioid receptor ligands, we considered that this residue parti- cipates in binding with Tyr1 of EM2, because the pres- ence 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 + or phe- hydrogen bond between the Tyr1 amino NH3 nolic 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 suit- able 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 several docking over the folded conformer. Thus, models were constructed for the open conformers and subjected to energy evaluation for reasonable configur- ation and translation exploration using the Docking module of insight discover. Consequently, it was suggested that the pocket shape and size of the model receptor 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)O...HO(Tyr- 148) (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 stabil- ization 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.
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Fig. 8. Stereoscopic superimpositions of EM2OH (green, molecules A and B), D-TIPP- NH2 (red, molecules A and B) and [Chx2]EM2 (black). The backbone chains are represented by thick bonds, and the intra- molecular hydrogen bonds are shown by dotted lines.
Y. In et al. Conformational comparison of endomorphin-2 and its C-terminal free acid
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 con- formation 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 pro- vide a structural–conformational reason why the C-ter- minal amidation of EM is necessary for its l-opioid receptor agonist activity.
Fig. 9. Stereoscopic view of possible dock- ing 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 resi- dues of the receptor for the interaction are shown by a ball and stick model. The hydro- gen bonds or electrostatic interactions are represented by dotted lines.
Experimental procedures
Peptides
ously [22]. For the binding assay, synaptosomal brain mem- brane P2 preparations from Sprague–Dawley rats were used as sources of l- and d-opioid receptors after the removal of endogenous opioids. The competitive displace- ment assay used 3.5 nm [3H]Tyr-d-Ala-Gly-MePhe-Gly-ol and 5.57 nm [3H][d-penicillamine 2-d-penicillamine 5]enke- phalin for the l- and d-sites, respectively, and the affinity constants (Ki) were determined using a conventional proce- dure. 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 aer- ated (95% O2, 5% CO2, v ⁄ v) Krebs–Henseleit solution at 35 (cid:1)C. The tissue was stimulated transmurally between plat- inum 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, modi- fied Mg2+-free Krebs solution containing 0.1 mm ascorbic acid and 0.027 mm EDTA4Na at 35 (cid:1)C. The tissue was sti- mulated transmurally with trains of rectilinear pulses of 1 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.
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.
NMR measurements
Opioid activity measurements
1H-NMR spectra were recorded on a Varian unity INO- VA500 spectrometer with a variable temperature-control unit. EM2 or EM2OH was dissolved in 0.7 mL of TFE
The binding assays and in vitro bioassays of EM1, EM2, EM1OH and EM2OH were performed as described previ-
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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.
TSP-d4
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 concen- trations 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 (2,2,3,3-tetradeuterio-3-(trimethylsilyl)- internal propionic acid sodium salt). The solutions were degassed and sealed under vacuum. All NMR measurements were performed at 25 (cid:1)C. The temperature dependence of the chemical shift of each NH proton was measured in the range of 20–60 (cid:1)C (10 (cid:1)C intervals).
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 (1.6–3.5 A˚ ) and weak (1.6–5.0 A˚ ). Since the aromatic pro- 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 ⁄ 6 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 treat- ment was used. Other potential functions were all cal- from insight ii ⁄ culated according to the protocol 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
The NMR measurements were performed by the same procedure used in dimethyl sulfoxide [6]. Two-dimensional COSY, TOCSY and ROESY were acquired in the phase- sensitive mode using standard pulse programs available in the Varian software library. Continuous low-power irradi- ation 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 inten- sities were classified into three groups (strong, medium, and weak) to estimate proton-proton distance.
using
constants
equations
the
A possible torsion angle was estimated from the vicinal 3JHNCaH ¼ coupling 9.8cos2h–1.1cosh +0.4sin2h, where / ¼ |h–60|(cid:1) for the / torsion angle around the C¢i-1–Ni–Cai–C¢i bond sequence [23] and JHCaCbH ¼ 11.0cos2h)1.4cosh +1.6sin2h for the h angle around the H–Cai–Cbi–H bond [24].
Computational molecular modelling calculation
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: C32H36N4O6Æ3.5H2O, Mr ¼ 635.70, monoclinic, P21, a ¼ 19.687(2) A˚ , b ¼ 6.5058(7) A˚ , c ¼ 25.869(3) A˚ , b ¼ 101.370(2)(cid:1), V ¼ 3248.3(6) A˚ 3, Z ¼ 4, 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.
(Frepul), chirality (Fchiral),
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 mini- mum of the target function consisting of force constants for the covalent bond (Fcovalent), repulsive van der Waals’ contact 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 cal- culation to avoid the swapping the chiralities at the high
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 Crystallo- graphy [29]. The positional parameters of non-H atoms were refined by a full-matrix least-squares method with an- isotropic 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 isotro- pic displacement parameters (Uiso ¼ 1.2 Ueq for the asso- ciated 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
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Y. In et al. Conformational comparison of endomorphin-2 and its C-terminal free acid
of Z-Gly-Phe-NH2, Tyr-Lys-NH2, and Asp-Phe-NH2. Chem Pharm Bull 48, 374–381.
5 In Y, Fujii M, Sasada Y & Ishida T (2001) Structural
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
studies on C-amidated amino acids and peptides: Crys- tal structures of hydrochloride salts of C-amidated Ile, Val, Thr, Ser, Met, Trp, Gln and Arg, and comparison with their C-unamidated ones. Acta Cryst B57, 72–81. 6 In Y, Minoura K, Ohishi H, Minakata H, Kamigauchi M, Sugiura M & Ishida T (2001) Conformational com- parison of l–selective endomorphin-2 with its C-ter- minal free acid in DMSO solution, by 1H NMR spectroscopy and molecular modeling calculation. J Peptide Res 58, 399–412.
7 Zadina JE, Hackler L, Ge LJ & Kastin AJ (1997) A
potent and selective endogenous agonist for the l-opioid receptor. Nature 386, 499–502.
8 Chang KJ, Lillian A, Hazum E, Cuatrecasas P & Chang
JK (1981) Morphiceptin (NH4-Tyr-Pro-Phe-Pro- CONH2): a potent and specific agonist for morphine (l) receptors. Science 212, 75–77.
9 Schiller PW, Nguyen TMD, Weltrowska G, Wilkes BC, Marsden. J, Lemieux C & Chung NN (1992) Differen- tial stereochemical requirements of l vs. d opioid receptors for ligand binding and signal transduction: Development of a class of potent and highly d-selective peptide antagonists. Proc Natl Acad Sci USA 89, 11871–11875.
10 Schwyzer R (1977) ACTH: a short introductory review.
Ann N Y Acad Sci 297, 3–26.
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 mod- elling [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 Pro- tein 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 per- formed on a graphics computer while keeping the confor- mation, and then the possible docking mode was refined using the Docking module of insight discover, where the energy evaluation was performed for the reasonable confi- guration and translation exploration.
Y. In et al. Conformational comparison of endomorphin-2 and its C-terminal free acid
Acknowledgements
11 In Y, Minoura K & Ishida T (2005) Conformational comparison of l-selective endomorphin-2 with its C-terminal free acid, studied by 1H-NMR spectroscopy and X-ray structure analysis. Peptide Sci 200, 427– 430.
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Cul- ture, Sports, Sciences, and Technology of Japan (Y.I.).
12 Al-Khrasani M, Orosz G, Kocsis L, Farkas V, Magyar A, Lengyel I, Benyhe S, Borsodi A & Ronai AZ (2001) Receptor constants for endomorphin-1 and endomor- phin-1-ol indicate differences in efficacy and receptor occupancy. Eur J Pharmacol 421, 61–67.
13 Khaled MA, Long MM, Thompson WD, Bradley RJ,
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The following material 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.
25 Clore GM, Nigles M, Sukumaran DK, Bruenger AT, Karplus M & Gronenborn AM (1986) The three-
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