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
1
H-NMR spectroscopy, molecular
calculation, and X-ray crystallography
Yasuko In
1
, Katsuhiko Minoura
1
, Koji Tomoo
1
, Yusuke Sasaki
2
, Lawrence H. Lazarus
3
,
Yoshio Okada
4
and Toshimasa Ishida
1
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:
(Received 30 June 2005, revised 8 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-NH
2
), 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
1
H-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
bond of (Tyr1)NH
3
+
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-NH
2
, Tyr-Tic-Phe-Phe-NH
2
; TSP-d
4
, 2,2,3,3-tetradeuterio-3-(trimethylsilyl)propionic acid sodium salt.
FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS 5079
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.
N-Terminal amidated endomorphin-1 (EM1, Tyr-
Pro-Trp-Phe-NH
2
) and endomorphin-2 (EM2, Tyr-
Pro-Phe-Phe-NH
2
) 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-NH
2
)
and its C-terminal free acid [8]. Furthermore, the
d-opioid receptor selectivity of the l-opioid receptor-
specific agonist TIPP-NH
2
(Tyr-Tic-Phe-Phe-NH
2
,
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.
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-NH
2
, where the
important feature for the opioid activity is the presence
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
1
H-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
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
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.
Conformational comparison of endomorphin-2 and its C-terminal free acid Y. In et al.
5080 FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS
pharmacological activities of these compounds as
l- and d-opioid receptor agonists are given in
Table 1. 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 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-
Pro-NH
2
) and its C-terminal free acid [8]. The K
i

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
agonist TIPP-NH
2
(Tyr-Tic-Phe-Phe-NH
2
) was
increased significantly if the C-terminal amide was
replaced by a free acid. It is obvious from the pre-
sent results that the differentiation between the
l- and d-opioid receptor selectivities of EM2 is rela-
ted 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 2.
1
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 diffi-

cult for these aromatic protons. Because of the
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 C aH ⁄ CdH
protons, and the population ratio determined by the
comparison of the proton peak intensities is given in
Table 2.
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).
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 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
Table 1. Binding affinities and the pharmacological activities of EM1, EM2, EM1OH and EM2OH for l-andd-opioid receptors.
Compound
Receptor binding In vitro agonist bioassay
l-Opioid
receptor K
i
(nM)
d-Opioid
receptor K
i
(nM)
Guinea pig ileum assay
(l-opioid receptor) IC
50
(nM)
Mouse vas deferens assay
(d-opioid receptor) IC
50

(nM)
EM1 0.36 1500 10.1 ± 1.2 36.3 ± 5.2
EM1OH 200 1800 4032 ± 4330 > 10
4
EM2 0.69 9200 5.79 ± 0.4 344 ± 93
EM2OH 200 3950 > 10
4
>10
4
Y. In et al. Conformational comparison of endomorphin-2 and its C-terminal free acid
FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS 5081
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).
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,
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
Table 2. Summary of the overall conformational characteristics of EM2 and EM2OH in DMSO, TFE, H

2
O (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.
Solvent
electronic form DMSO TEF
H
2
O DPC
pH 2.7 pH 5.2 pH 3.5 pH 5.2
EM2 trans ⁄ cis ¼ 2:1 trans ⁄ cis ¼ 3:2 trans ⁄ cis ¼ 3:2 trans
trans Neutral Open (rS ¼ 30) Open (rS ¼ 18,
n7 ¼ 4)
fold (F2 ¼ 8)
––––
Monocation – – 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 – – Open (rS ¼ 7, n7 ¼ 20)
(F2 ¼ 2)
––
EM2OH trans ⁄ cis ¼ 2:1 trans ⁄ cis ¼ 1:1 trans ⁄ cis ¼ 3:2 trans ⁄ cis ¼ 1:1 trans trans ⁄ cis ¼ 2:1
trans Zwitter Open (rS ¼ 6,
n7 ¼ 12)
fold (F1 ¼ 7,

F2 ¼ 5)
Open (n7 ¼ 11)
fold (F1 ¼ 14,
F2 ¼ 5)
Open (rS ¼ 4,
n7 ¼ 14)
fold (F1 ¼ 8,
F2 ¼ 4)
Open (rS ¼ 21,
n7 ¼ 2)
fold (F2 ¼ 7)
Open (rS ¼ 12,
n7 ¼ 9)
fold (F1 ¼ 1,
F2 ¼ 8)
Fold (F1 ¼ 30)
Monocation – – Open (rS ¼ 18,
n7 ¼ 10)
fold (F2 ¼ 2)
–––
cis Zwitter Fold (F1 ¼ 30) Fold (F1 ¼ 30) Open (rS ¼ 16,
n7 ¼ 2)
fold (F1 ¼ 12)
Open (rS ¼ 5,
n7 ¼ 7)
fold (F1 ¼ 18)
– Fold (F1 ¼ 30)
Monocation – – Open (rS ¼ 30) – – –
Conformational comparison of endomorphin-2 and its C-terminal free acid Y. In et al.
5082 FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS

considered: for EM2 in neutral form (N-terminal
NH
2
and C-terminal NH
2
) in TFE and in monocati-
onic form (N-terminal NH
3
+
and C-terminal NH
2
)
in water and DPC micelles; for EM2OH in zwitteri-
onic form (N-terminal NH
3
+
and C-terminal COO
–
)
in TFE, water and DPC micelles and in monocation-
ic form (N-terminal NH
3
+
and C-terminal COOH)
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
folding pathway, in which the target function was
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°. 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-
minal polar atoms, that is, their hydrogen-bonded
(F1-type) and nonhydrogen-bonded (F2-type) fol-
dings. The results are given in Table 2.
Conformational characteristics of EM2
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
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
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 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
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 (–).
Residue TFE
H
2
O DPC
pH 2.7 pH 5.2 pH 3.5 pH 5.2
EM2
trans
Phe3NH 4.06 9.76 9.76 8.47 8.47
Phe4NH 3.32 6.68 6.68 7.32 7.32
C-term.NH
2
3.40 0.60 0.60 – _
4.29 3.90 3.90 – –
cis
Phe3NH 4.58 7.70 7.70
Phe4NH 4.20 7.62 7.62
C-term.NH
2
3.01 – –
5.11 – –
EM2OH

trans
Phe3NH 3.27 9.49 8.63 7.13 7.67
Phe4NH 5.68 6.35 3.00 6.56 4.46
cis
Phe3NH 3.93 7.81 7.18 6.47
Phe4NH 6.10 7.34 4.88 2.01
Y. In et al. Conformational comparison of endomorphin-2 and its C-terminal free acid
FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS 5083
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
TFE
trans EM2 Tyr1 b2 Pro2 d1 Weak trans EM2OH Tyr1 b2 Phe3 NH Weak
Phe4 a C-NH2 Medium
cis EM2 Phe4 a C-NH2 Medium cis EM2OH Tyr1 a Phe3 NH Weak
Tyr1 2,6H Pro2 d1 Weak
Tyr1 3,5H Pro2 a Weak
Phe3 b2 Phe4 NH Weak
H
2
O (pH 2.7)
trans EM2 Tyr1 b1 Pro2 d2 Weak trans EM2OH Tyr1 2,6H Pro2 d2 Weak
Phe3 3,5H Phe4 a Weak Phe3 2,6H Phe4 NH Medium

Phe4 a C-NH1 Medium
Phe4 a C-NH2 Weak
cis EM2 Tyr1 b1 Pro2 a Medium cis EM2OH Tyr1 b2 Phe3 2,6H Medium
Tyr1 b2 Pro2 a Strong Tyr1 2,6H Pro2 d1 Weak
Pro2 c2 Phe3 NH Weak Pro2 a Phe4 NH Medium
Phe3 a Phe4 NH Strong
Phe3 b2 Phe4 NH Medium
H
2
O (pH 5.2)
trans EM2 Tyr1 b1 Pro2 d2 Weak trans EM2OH Tyr1 3,5H Pro2 c Medium
Phe3 3,5H Phe4 a Weak Pro2 b1 Phe4 NH Weak
Phe3 b1 Phe4 NH Weak Phe3 NH Phe4 NH Weak
Phe4 a C-NH1 Medium
Phe4 a C-NH2 Weak
cis EM2 Pro2 b2 Phe3 NH Weak cis EM2OH Tyr1 3,5H Pro2 a Medium
Pro2 c2 Phe3 NH Weak Pro2 b1 Phe4 NH Weak
Phe3 NH Phe4 NH Weak
Phe3 a Phe4 NH Medium
Phe3 b2 Phe4 NH Medium
DPC (pH 3.5)
trans EM2 Tyr1 2,6H Phe3 a Weak trans EM2OH Pro2 b1 Phe4 2,6H Weak
Pro2 b1 Phe3 2,6H Weak Pro2 b1 Phe4 3,5H Weak
Phe3 b2 Phe4 2,6H Weak Pro2 c1 Phe4 2,6H Weak
Phe3 2,6H Phe4 2,6H Medium Pro2 c1 Phe4 3,5H Weak
Phe3 2,6H Phe4 3,5H Medium Phe3 a Phe4 b2 Weak
Phe3 3,5H Phe4 a Medium Phe3 a Phe4 2,6H Medium
Phe3 3,5H Phe4 2,6H Medium Phe3 b2 Phe4 NH Weak
DPC (pH 5.2)
trans EM2 Tyr1 2,6H Phe3 a Weak trans EM2OH Pro2 b1 Phe3 NH Weak

Phe3 2,6H Phe4 a Weak Pro2 c1 Phe3 2,6H Weak
Phe3 2,6H Phe4 3,5H Medium Phe3 b1 Phe4 2,6H Weak
Phe3 2,6H Phe4 2,6H Medium Phe3 b2 Phe4 NH Weak
Phe3 3,5H Phe4 a Medium
Phe3 3,5H Phe4 2,6H Medium cis EM2OH Tyr1 a Pro2 a Medium
Tyr1 2,6H Pro2 a Medium
Tyr1 2,6H Pro2 d1 Weak
Tyr1 3,5H Pro2 a Medium
Pro2 a Phe3 NH Medium
Phe3 NH Phe4 NH Weak
Phe3 a Phe4 NH Weak
Phe3 b1 Phe4 2,6H Medium
Phe3 b2 Phe4 2,6H Weak
Conformational comparison of endomorphin-2 and its C-terminal free acid Y. In et al.
5084 FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS
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.
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 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 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 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.
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
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
A
B
C
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
FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS 5085
existed almost completely as a trans rotamer, similar
to the case of trans EM2.
Trans 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
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 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 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
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.
Conformational comparison of endomorphin-2 and its C-terminal free acid Y. In et al.
5086 FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS
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 con-
formers (conformers A and B) and seven water mole-
cules per asymmetric unit. These conformers are
shown in Fig. 7. Selected conformational torsion
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
mainly stabilized by an intramolecular (Tyr1)NH
3
+

–

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
water molecule (O6W) with NH
3
+
(Tyr1), NH(Phe3)
and
–
OOC(Phe4), in addition to the indirect interac-
tion between both terminal polar groups via a water
molecule (O7W): a O7W NH
3
+
(Tyr1) electrostatic
interaction and a O7W OOC
–
(Phe4) hydrogen bond.
A
B
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.
Y. In et al. Conformational comparison of endomorphin-2 and its C-terminal free acid
FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS 5087
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 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
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.
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.
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,
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.
Conformational comparison of endomorphin-2 and its C-terminal free acid Y. In et al.
5088 FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS
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
bond) between the N-terminal NH
3

+
and C-terminal
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.
AB
CD
EF
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
FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS 5089
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-
formation despite environmental changes, whereas

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
opioid peptide, because it provides well-defined
Fig. 6. 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 back-
bone chains.
Conformational comparison of endomorphin-2 and its C-terminal free acid Y. In et al.
5090 FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS
molecular conformations at the atomic level. As for
the l-opioid receptor-specific agonists, two different
crystals have been reported, i.e. d-TIPP-NH
2
(Tyr-d-
Tic-Phe-Phe-NH
2
) [17] and [Chx2]EM2 (Tyr-Chx-Phe-
Phe-NH
2
) [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
(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-NH
2
,
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-NH
2
. On the other hand,
the conformation of [Chx2]EM2 did not form such
a C-terminal NH

2
-participating hydrogen 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
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
FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS 5091
Table 5. Torsion angles of cis EM2OH conformers.
Conformer /wxv1 v2 v3 v4 v5
Tyr1 A 140.7(4) 4.7(4) 169.8(4) )85.6(4)
B 134.1(4) 8.0(4) )176.7(4) )96.4(4)
Pro2 A )96.0(4) )1.5(4) 177.7(4) 36.5(4) )37.9(4) 24.0(4) )0.8(3) )22.2(3)
B )92.2(4) 9.4(4) 173.7(4) 37.8(3) )40.1(4) 26.3(3) )2.1(3) )22.2(3)
Phe3 A )109.3(4) )50.4(4) )176.1(4) )58.6(4) )80.0(4)
B )105.8(4) )139.6(4) 167.4(4) )56.8(4) )79.6(4)
Phe4 A )136.3(4) )20.1(4) )57.5(4) )73.9(5)
B )76.1(4) )32.5(4) )60.1(4) )95.8(5)
Table 6. Intermolecular hydrogen bonds and selected short contacts of cis EM2OH.
Donor at x,y,z D-H Acceptor A
Length (A
˚

)
Angle(°) < D-H A Symmetry operation of AD. A H. A
Hydrogen bonds
N(1)A O(4¢¢)A 2.728(5) 1.85 154 x,y,z
N(1)A O(4W) 2.848(5) 1.97 155 x +1,y,z
N(1)A O(7W) 2.754(5) 1.91 154 x +1,y,z
O(1H)A O(4¢¢)B 2.598(5) 1.66 157 1-x,y +1 ⁄ 2,-z
N(3)A O(2W) 2.899(5) 2.13 146 x,y,z
N(4)A O(2W) 2.936(5) 1.88 174 x,y,z
N(1)B O(5W) 2.825(5) 1.98 165 x,y,z
N(1)B O(6W) 2.857(5) 2.02 158 x,y,z
N(1)B O(3 W) 2.778(5) 1.97 148 x-1,y,z
O(1H)B O(4¢)A 2.592(5) 1.75 139 1-x,y-1 ⁄ 2,1-z
N(3)B O(6W) 2.968(5) 2.28 139 x,y,z
O(1W) O(1¢)A 2.773(4) 1.76 167 x,y +1,z
O(1W) O(3 W) 2.871(4) 1.98 148 x,y +1,z
O(2W) O(3¢)A 2.756(5) 1.82 167 x,y-1,z
O(2W) O(1W) 2.822(4) 1.94 173 x,y-1,z
O(3W) O(4¢¢)A 2.682(5) 1.86 172 x,y,z
O(3W) O(1H)B 2.981(5) 2.07 162 1-x,y-1 ⁄ 2,1-z
O(4W) O(5 W) 2.796(4) 2.04 138 x,y,z
O(4W) O(1H)A 3.016(4) 2.19 145 1-x,y +1 ⁄ 2,-z
O(5W) O(1¢)B 2.791(5) 1.93 164 x,y +1,z
O(6W) O(4¢¢)B 2.765(5) 2.01 156 x,y,z
O(6W) O(3¢)B 2.886(5) 2.16 146 x,y-1,z
O(7W) O(4¢)B 2.760(5) 1.87 170 x,y,z
O(7W) O(4W) 2.831(4) 1.90 164 x,y-1,z
Electrostatic short contacts
N(1)A O(1W) 3.024(4) x,y,z
N(3)A N(2)A 2.783(5) x,y,z

O(1H)A O(4W) 3.016(4) 1-x,y-1 ⁄ 2,-z
O(1H)A O(4¢)B 3.138(5) 1-x,y-1 ⁄ 2,-z
N(1)B O(7W) 3.021(5) x,y,z
N(3)B N(2)B 2.776(5) x,y,z
O(2W) O(4¢¢)A 3.273(5) x,y,z
O(3W) O(4¢)A 3.122(5) x,y-1,z
O(4W) O(4¢)B 2.814(4) x,y,z
O(4W) N(1)A 2.848(5) x-1,y,z
O(4W) O(1W) 3.221(4) x-1,y,z
O(5W) O(4¢)A 2.767(5) x-1,y,z
O(6W) C(1¢)B 3.083(5) x,y,z
Conformational comparison of endomorphin-2 and its C-terminal free acid Y. In et al.
5092 FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS
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.
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 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
hydrogen bond between the Tyr1 amino NH
3
+

or phe-
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
over the folded conformer. Thus, several docking
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 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(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.
Fig. 8. Stereoscopic superimpositions of
EM2OH (green, molecules A and B),
D-TIPP-
NH
2
(red, molecules A and B) and
[Chx2]EM
2
(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
FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS 5093
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.
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.
Opioid activity measurements
The binding assays and in vitro bioassays of EM1, EM2,
EM1OH and EM2OH were performed as described previ-
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 [
3
H]Tyr-d-Ala-Gly-MePhe-Gly-ol
and 5.57 nm [
3
H][d-penicillamine 2-d-penicillamine 5]enke-
phalin for the l- and d-sites, respectively, and the affinity
constants (K
i
) 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% O
2
,5%CO
2,
v ⁄ v) Krebs–Henseleit solution at
35 °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 Mg
2+
-free Krebs solution containing 0.1 mm ascorbic
acid and 0.027 mm EDTA4Na at 35 °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 IC
50
values (concentration causing a 50%
reduction in the number of the electrically induced twitches)
were calculated.
NMR measurements
1

H-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
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.
Conformational comparison of endomorphin-2 and its C-terminal free acid Y. In et al.
5094 FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS
solvent, H
2
O ⁄ D
2
O (9 : 1), or DPC micelles, where the
40-fold excess molar ratio of perdeuterated DPC to EM2
or EM2OH is dissolved in H
2
O ⁄ D
2
O (9 : 1). The concen-
trations used for NMR measurements were 10 mm for
TFE, 16 mm for H
2
O ⁄ D
2

O, 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-d
4
(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 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.
A possible torsion angle was estimated from the vicinal
coupling constants using the equations

3
J
HNCaH
¼
9.8cos
2
h–1.1cosh +0.4sin
2
h, where / ¼ |h–60|° for the /
torsion angle around the C¢i-1–Ni–Cai–C¢i bond sequence
[23] and J
HCaCbH
¼ 11.0cos
2
h)1.4cosh +1.6sin
2
h 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 mini-
mum 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 cal-
culation to avoid the swapping the chiralities at the high
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
(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 (< r
6
>)
)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-
culated 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 mm
3
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: C
32
H
36
N
4
O
6
Æ3.5H
2
O, Mr ¼ 635.70,
monoclinic, P2
1

,a¼ 19.687(2) A
˚
, b ¼ 6.5058(7) A
˚
, c ¼
25.869(3) A
˚
, b ¼ 101.370(2)°, V ¼ 3248.3(6) A
˚
3
, Z ¼ 4,
F
000
¼ 1356, l(Mo Ca) ¼ 0.096 mm
)1
, number of observed
reflections ¼ 20743, R
int
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,
Dq
max
¼ 0.367 eA
˚
)3
, and Dq
min

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 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
Y. In et al. Conformational comparison of endomorphin-2 and its C-terminal free acid
FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS 5095
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 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.
Acknowledgements
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.).
References
1 Eipper BA & Mains RE (1988) Peptide alpha-amida-
tion. Annu Rev Physiol 50, 333–344.
2 Yang YR, Chiu TH & Chen CL (1999) Structure-activ-
ity relationships of naturally occurring and synthetic
opioid tetrapeptides acting on locus coeruleus neurons.

Eur J Pharmacol 372, 229–236.
3 Gentilucci L & Tolomelli A (2004) Recent advances in
the investigation of the bioactive conformation of pep-
tides active at the l–opioid receptor. Conformational
analysis of endomorphins. Curr Topics Med Chem 4,
105–121.
4 In Y, Tani S & Ishida T (1999) Structural studies of
C-amidated amino acids and peptides: Crystal structures
of Z-Gly-Phe-NH
2
, Tyr-Lys-NH
2
, and Asp-Phe-NH
2
.
Chem Pharm Bull 48, 374–381.
5 In Y, Fujii M, Sasada Y & Ishida T (2001) Structural
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
1
H 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 (NH
4
-Tyr-Pro-Phe-Pro-
CONH
2
): 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.
11 In Y, Minoura K & Ishida T (2005) Conformational
comparison of l-selective endomorphin-2 with its
C-terminal free acid, studied by
1
H-NMR spectroscopy
and X-ray structure analysis. Peptide Sci 200, 427–
430.
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,
Brown GB & Urry DW (1977) Conformational states of
enkephalins in solution. Biochem Biophys Res Commun
76, 224–231.
14 Zetta L & Cabassi F (1982) 270-MHz
1
H nuclear-
magnetic-resonance study of met-enkephalin in solvent
mixtures. Conformational transition from dimethyl-
sulphoxide to water. Eur J Biochem 122, 215–222.
15 Fiori S, Renner C, Cramer J, Pegoraro S & Moroder L
(1999) Preferred conformation of endomorphin-1 in
aqueous and membrane-mimetic environments. J Mol
Biol 291, 163–175.
16 Podlogar BL, Paterlini G, Ferguson DM, Leo GC,
Demeter DA, Brown FK & Reitz AB (1998) Conforma-
tional analysis of the endogenous l–opioid agonist
Conformational comparison of endomorphin-2 and its C-terminal free acid Y. In et al.
5096 FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS
endomorphin-1 using NMR spectroscopy and molecular
modeling. FEBS Lett 439, 13–20.
17 Flippen-Anderson JL, Deschamps JR, George C, Reddy
PA, Lewin AH, Brine GA, Sheldrick G & Nikiforovich
G (1997) X-ray structure of Tyr-D-Tic-Phe-Phe-NH
2
(D-TIPP-NH
2
), a highly potent l–receptor selective
opioid agonist: comparison with proposed model

structures. J Peptide Res 49, 384–393.
18 Doi M, Asano A, Komura E & Ueda Y (2002) The
structure of an endomorphin analogue incorporating
1-aminocyclohexane-1-carboxylic acid for proline is
similar to the b–turn of Leu-enkephalin. Biochem
Biophys Res Commun 297, 138–142.
19 Lengyel I, Orosz G, Biyashev D, Kocsis L, Al-Khrasani
M, Ronai A, Tomboly Cs, FurstZs Toth G & Borsodi
A (2002) Side chain modifications change the binding
and agonist properties of endomorphin 2. Biochem
Biophys Res Commun 290, 153–161.
20 Mosberg HI & Fowler CB (2002) Development and
validation of opioid ligand–receptor interaction models:
The structural basis of mu vs. delta selectivity. J Peptide
Res 60, 329–335.
21 Mansour A, Taylor LP, Fine JL, Thompson RC, Hov-
ersten 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.
22 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 endomor-
phin-2 analogues with mixed l- ⁄ d-opioid agonist and
d-opioid antagonist properties. J Med Chem 47, 3591–
3599.
23 Bystrov VF (1976) Spin-spin coupling and the conform-
ational states of peptide systems. Prog Nucl Magn Reson
Spectrosc 10, 41–81.

24 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.
25 Clore GM, Nigles M, Sukumaran DK, Bruenger AT,
Karplus M & Gronenborn AM (1986) The three-
dimensional structure of a1-purothionin in solution:
combined use of nuclear magnetic resonance, distance
geometry and restrained molecular dynamics. EMBO
J 5, 2729–2735.
26 Nilges M, Clore GM & Gronenborn AM (1988) Deter-
mination of three-dimensional structures of proteins
from interproton distance data by hybrid distance geo-
metry-dynamical simulated annealing calculations.
FEBS Lett 229, 317–324.
27 INSIGHT ⁄ DISCOVER. Accelrys Software Inc, San
Diego, CA.
28 Sheldrick GM (1997) SHELXS97. Program for the
Refinement of Crystal Structures. University of Gottin-
gen, Germany.
29 Edited by Theo Hahn (1992) International Tables for
X-Ray Crystallography. Vol. C Kluwer Academic
Publishers, Dordrecht.
30 Sheldrick GM (1997) SHELXL97. Program for the
Solution of Crystal Structure. University of Gottingen,
Germany.
31 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.
Y. In et al. Conformational comparison of endomorphin-2 and its C-terminal free acid
FEBS Journal 272 (2005) 5079–5097 ª 2005 FEBS 5097