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

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

Conformational comparison of l-selective endomorphin-2 with its

calculation, and X-ray crystallography

Yasuko In1, Katsuhiko Minoura1, Koji Tomoo1, Yusuke Sasaki2, Lawrence H Lazarus3,

Yoshio Okada4and 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)

doi:10.1111/j.1742-4658.2005.04919.x

To investigate the structural function of the C-terminal amide group of endomorphin-2 (EM2, H-Tyr-Pro-Phe-Phe-NH2), an endogenous l-opioid receptor ligand, the solution conformations of EM2 and its C-terminal free acid (EM2OH, H-Tyr-Pro-Phe-Phe-OH) in TFE (trifluoroethanol), water (pH 2.7 and 5.2), and aqueous DPC (dodecylphosphocholine) micelles (pH 3.5 and 5.2) were investigated by the combination of 2D 1H-NMR 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)NH3+ COO–(Phe4), observed in its crystal structure These results show that the substitution of a carboxyl group for the C-terminal

Abbreviations

EM1, endomorphin-1; EM2, endomorphin-2; EM2OH, C-terminal free acid endomorphin-2; Tic, tetrahydro-3-isoquinoline carboxylic acid; TIPP-NH 2 , Tyr-Tic-Phe-Phe-NH 2 ; TSP-d 4 , 2,2,3,3-tetradeuterio-3-(trimethylsilyl)propionic acid sodium salt.

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-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 free acid [8] Furthermore, the

d-opioid receptor selectivity of the l-opioid

receptor-specific agonist TIPP-NH2 (Tyr-Tic-Phe-Phe-NH2,

where Tic¼ 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-NH2, 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 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 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

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

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-NH2) and its C-terminal free acid [8] The Ki

values for the binding affinity also suggest that the

d-opioid receptor affinity of EM2 is increased by the

substitution of a carboxyl group for the C-terminal

amide group, and a similar phenomenon has been

reported by Schiller et al [9], where the d-opioid

receptor selectivity of the l-opioid receptor-specific

agonist TIPP-NH2 (Tyr-Tic-Phe-Phe-NH2) 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

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-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 CaH⁄ CdH protons, and the population ratio determined by the comparison of the proton peak intensities is given in Table 2

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- and d-opioid receptors.

Compound

Receptor binding In vitro agonist bioassay

l-Opioid receptor Ki(n M )

d-Opioid receptor Ki(n M )

Guinea pig ileum assay (l-opioid receptor) IC50(n M )

Mouse vas deferens assay (d-opioid receptor) IC50(n M )

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

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, 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.

Solvent

electronic form DMSO TEF

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)

considered: for EM2 in neutral form (N-terminal

NH2 and C-terminal NH2) in TFE and in

monocati-onic form (N-terminal NH3+ and C-terminal NH2)

in water and DPC micelles; for EM2OH in

zwitteri-onic form (N-terminal NH3+ and C-terminal COO–)

in TFE, water and DPC micelles and in

monocation-ic form (N-terminal NH3+ 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)C¼ 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 < TFE¼ 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

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.NH2 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.NH2 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

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

H2O (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

H2O (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

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.

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 concon-former in water, (C) F1-type and (D) F2-type folded concon-formers in DPC micelles.

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 (concon-formers 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)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 water molecule (O6W) with NH3+(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 NH3+(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.

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

transrotamer 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 O¼ 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 acidmonocation-ic 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.