(196â228) with Opioid Peptide Dynorphin in ... - ACS Publications

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Interaction of Extracellular Loop II of κ‑Opioid Receptor (196−228) with Opioid Peptide Dynorphin in Membrane Environments as Revealed by Solid State Nuclear Magnetic Resonance, Quartz Crystal Microbalance and Molecular Dynamics Simulation Atsushi Kira,†,† Namsrai Javkhlantugs,†,‡ Takenori Miyamori,† Yoshiyuki Sasaki,† Masayuki Eguchi,† Izuru Kawamura,† Kazuyoshi Ueda,† and Akira Naito*,† †

Graduate School of Engineering, Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan Center for Nanoscience and Nanotechnology & School of Engineering and Applied Science, National University of Mongolia, Ulaanbaatar 14201, Mongolia

‡

ABSTRACT: κ-Opioid receptor is a member of the opioid receptor family and selectively interacts with the opioid peptide dynorphin. Extracellular loop II (ECL-II) of the κ-opioid receptor displays an amphiphilic helix in membrane environments and the N-terminal α-helix of dynorphin A(1−17) (hereafter DynA17) is inserted into the membrane with the tilt angle of 21° to the bilayer normal. ECL-II peptides (1−33), corresponding to 196−228 of κ-opioid receptor with [1-13C]- or [3-13C]-labeled amino acids were incorporated into large [dimyristoylphosphatidyl choline (DMPC)/ dihexanoylphosphatidyl choline (DHPC) = 3, q = 3] and small bicelle (q = 1) systems. 13 C direct detection with dipolar decoupling and magic angle spinning (DD-MAS) nuclear magnetic resonance (NMR) spectra were recorded, and the 13C chemical shift perturbation clearly indicated that DynA17 interacts with ECL-II at the location of Val10−Ala15. Quartz crystal microbalance measurements were performed to determine the binding constant of ECL-II with DynA17 and indicated that the binding constant between DynA17 and ECL-II embedded in the lipid layer was 72 times larger than that between DynA17 and the lipid. The result of the molecular dynamics simulation clearly indicates that the C-terminus of DynA17 interact with the amino acid residues of the region between Val10-Gln14 of ECL-II. These results suggest that DynA17 interacts with the ECL-II of the κ-opioid receptor through a hydrophobic and short-lived electrostatic interaction with high affinity in the outer surface of the membrane.

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INTRODUCTION The κ-, μ-, and δ-opioid receptors belong to the G-proteincoupled receptor superfamily, which are integral membrane proteins presumed to have the common following structural motifs, seven transmembrane (TM) helices, connecting loops, and long N- and C-terminal tails at the extracellular and intracellular domains. Studies involving κ-opioid receptor chimeras have shown that the putative second extracellular loop (ECL-II) contains eight amino acid residues and contributes substantially to the κ-opioid receptor’s selectivity in dynorphin (Dyn) ligand recognition.1−3 In comparison, dynorphin A(1−13) (DynA13), contains five basic amino acid residues (from Arg6 to Lys13). Removal or substitution with Ala of the basic residues in DynA13 causes a marked decrease in κ-receptor selectivity, especially Arg7.4,5 Since residues 12− 17 in dynorphin A(1−17) (DynA17) can be truncated without a significant loss of bioactivity,6 it is therefore reasonable to speculate that the DynA17 binds to the κ-opioid receptor through Columbic interactions. However, other investigators have performed docking studies based on a homology model of the κ-opioid receptor and have proposed that a helical domain in ECL-II interacts with dynorphin A(1−10) (DynA10) through hydrophobic interactions.7 © 2014 American Chemical Society

To explore the structural features contributing to the specific binding of the κ-receptor ECL-II domain to its selective analogues, a 33-amino acid peptide composed of a sequence based on residue 196−228 of the human κ-opioid receptor ECL-II sequence was synthesized.8 In addition to the amino acid residues believed to constitute the ECL-II domain of the receptor, this peptide included N-terminal and C-terminal amino acids believed to reside, respectively, in the transmembrane IV and V domains of the κ-receptor. C210 in the ECL-II peptide was replaced with A210, to increase the stability of the synthetic peptide. Furthermore, the N-terminus was acetylated, and the C-terminus was amidated. The amino acid sequence of ECL-II is as follows: Ac-Leu196(1)-Gly-Gly-Thr-LysVal-Arg-Glu-Asp-Val-Asp-Val-Ile-Glu-Ala-Ser-Leu-Gln-PhePro-Asp-Asp-Asp-Tyr-Ser-Trp-Trp-Asp-Leu-Phe-Met-LysIle228(33)-NH2. The structures deduced from distance geometry and simulated annealing present a family of structures that display an α-helical array from V6 to A15 with an RMSD of 0.48 Å for Received: June 2, 2014 Revised: July 16, 2014 Published: July 24, 2014 9604

dx.doi.org/10.1021/jp505412j | J. Phys. Chem. B 2014, 118, 9604−9612

The Journal of Physical Chemistry B

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the backbone atoms. The peptide is relatively undefined in the central hinge region because the S16−P20 sequence yields few nuclear Overhauser effects (NOEs). Following D21 is a β-turn spanning three consecutive Asp residues (D21−D23) and Y24. The C-terminus of the peptide displays a helical tendency, and the residues following V6 form a turn of approximately 90° from the N-terminus.8 The binding profile of the κ-opioid receptor is relatively unique among the opioid receptors, while those of the μ- and δopioid receptors are relatively similar. NMR data show that the κ-opioid receptor ECL-II domain displays an amphiphilic helical region from V201 to A210 (C210 in the original sequence), which is not present in the μ- and δ-opioid receptors. This observation supports the contention that the ECL-II amphiphilic helical domain interacts with DynA10 based on sequence analysis and homology modeling of the κopioid receptor. Importantly, an amphiphilic helical region is present in DynA17 from residue Gly3 to Arg9, which was previously studied in a DPC micelle environment,9 and the sequence of DynA17 is Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-ArgPro-Lys-Leu-Lys-Trp-Asp-Asn-Gln-OH. The structure and orientation of DynA17 bound to lipid bilayers have recently been investigated by our group.10,11 It was found that DynA17 adopts an α-helical structure in the Nterminus from Gly2 to Leu5. In contrast, it adopts a disordered conformation from the center to the C-terminus and is located on the membrane surface. It was revealed that the N-terminal α-helix is inserted into the membrane with a tilt angle of 21° with respect to the bilayer normal. The complementary helix−helix binding mode is a reasonable proposal for the binding of endogenous dynorphin.11 Moreover, the helix−helix interaction is suggested to be hydrophobic in nature because the binding of DynA13 is essentially not affected by charge neutralization of the ECL-II. A β-turn around the D22 (D217) and D23 (D218) residues represents another feature of the ECL-II. The side chains of these two aspartic acids are exposed to the aqueous medium, and the significance of this β-turn remains undetermined. Similarly, the nociception (Noc) receptor ECL-II domain has been proposed to be involved in ligand binding and selectivity. This complex is stabilized by ionic interaction between the two Noc basic motifs and the ECL-II acidic residues, namely hydrophobic contacts involving the Noc FGGF N-terminal sequence and an ECL-II tryptophan residue.12 Recently, the crystal structure of human κ-opioid receptorT4L, in complexed with JDTic was reported.13 ECL-II of the human κ-opioid receptor forms a β-hairpin rather than an αhelix, as described earlier. The structure reveals the ligand binding pocket to be comparatively large and partially coupled with the ECL-II β-hairpin. However, the interaction of ECL-II with ligand molecules was not discussed. In this work, a detailed binding profile of the κ-receptor ECLII domain and DynA17 in a lipid bilayer environment is characterized by means of 13C solid-state nuclear magnetic resonance (NMR), quartz crystal microbalance (QCM), and molecular dynamics (MD) simulation analysis to gain insight into the mechanism of the formation of ECL-II/DynA17 complexes. Bicelle systems composed of dimyristoylphosphatidyl choline (DMPC) and dihexanoylphosphatidyl choline (DHPC) were used as a model of mammalian cell membranes.

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EXPERIMENTAL MATERIALS AND METHODS

Peptide Synthesis. ECL-II (κ-receptor, L196−I228) peptides, selectively 13C labeled at the carbonyl carbons of Gly3, Val6, Val10, Val12, Ile13, Leu17, or methyl carbon at Ala15, were synthesized, respectively, using a solid phase method with an Applied Biosystems 431A peptide synthesizer with an amide resin (Applied Biosystems, Inc., Foster City, California).14 After removing the protecting groups and cleavage from the resin, the synthesized peptides were purified using a Waters 600E high-performance liquid chromatography (HPLC) equipped with a Wako Navi C18−5 reversed-phase column. The purity of peptide was more than 95%, as estimated by the chromatogram. The synthetic ECL-II was further acetylated at the N-terminus and amidated at the C-terminus. Further, C210 was replaced with A210 to stabilize the peptide. Since this synthetic ECL-II was poorly soluble in aqueous solutions, a mixture of 40% citric acid and acetonitrile containing 0.05% of TFA was used as a mobile phase for HPLC. Purified ECL-II was incorporated into two types of bicelles with different lipid compositions, large bicelle (DMPC:DHPC = 3:1, q = [DMPC]/[DHPC] = 3) and small bicelle (DMPC:DHPC = 1:1, q = 1), where q indicates the molar ratio of DMPC to DHPC.15 Bicelle systems were used as a model of lipid bilayer, and thus, relatively large bicelles with q values of more than 1 were used in the interaction analysis. DynA17 interacts uniformly with ECL-II on the bilayer in both surfaces. This is a great advantage to use the bicelle system as compared with multilamellar vesicle systems in which only DynA17 interacts to ECL-II in the most outer surface.16,17 NMR Measurements. 31P and 13C NMR spectra were recorded on a Chemagnetics CMX 400 Infinity NMR spectrometer at the 31P and 13C resonance frequencies of 161.1 and 100.1 MHz, respectively, under static and magicangle spinning (MAS) conditions using the DD method (direct detection with high power proton decoupling).18 In the DD method, 5 μs of 90° pulse was applied to 13C or 31P nuclei followed by 50 kHz amplitude of 1H decoupling pulse with the duration time of 50 ms. MAS spinning frequency was set to 4 kHz. 1000 and 10000 Transients were accumulated for 31P DDstatic and 13C DD-MAS NMR spectra, respectively. In the process of Fourier transformation, 30 and 5 Hz of line broadening were applied for large and small bicleles, respectively. 31P and 13C chemical shift values were referred to those of 85% H3PO4 and tetramethylsilane (TMS), respectively. QCM Measurements. The quartz crystal microbalance (QCM) method was used for affinity and kinetics analysis of the interactions of DynA17 with DMPC lipid bilayer and ECLII. QCM measurements were performed on an AffinixQ4 (Initium Inc., Japan) with a 27-MHz AT-cut quartz resonator. The relationship between the changes in mass (Δm, g) and those in frequency (ΔF, Hz), as determined using the AT-cut QCM, follows Sauerbrey’s equation (eq 1):19 ΔF = −

2F02 Δm A ρq μq

(1)

where F0 is the original frequency of the QCM (27 × 106 Hz), A is the area of the gold electrode (4.9 × 10−2 cm2), ρq is the density of the quartz (2.65 g cm−3), and μq is the shear modulus of quartz (2.95 × 1011 dyn cm−2). In the case of the 27-MHz 9605



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AT-cut QCM, a decrease in frequency by 1 Hz corresponds to a 0.61 ng cm−2 increase in the mass of the gold electrode. After the QCM sensor was photochemically cleaned with UV−ozone, the gold electrode of the QCM sensor was modified with an alkanethiol self-assembly monolayer (SAM) of n-octadecanethiol. Modification of the QCM sensors with noctadecanethiol was carried out by immersing the sensors in a 1 mM ethanol solution of n-octadecanethiol for 24 h at room temperature followed by washing the sensors with ethanol and deionized water. DMPC and ECL-II reconstituted in DMPC were immobilized as a lipid monolayer on the gold electrode of the QCM sensor modified with alkanethiol SAM.20,21 ECL-II was reconstituted into DMPC at several molar ratios. The small unilamellar vesicles (SUVs) of DMPC and ECL-II reconstituted into DMPC were prepared under sonication from multilamellar vesicle (MLV), according to our previous paper.22 QCM measurements were carried out at 25 °C in 500 μL of Tris buffer (pH 7.4) containing 138 mM NaCl and 2.7 mM KCl, and DynA17 was injected into the QCM sensors covered by lipid bilayers. Computational Procedure. Molecular dynamics (MD) simulation was performed using CHARMM3423 with all-atom force field24−26 to investigate the interaction of DynA17 with ECL-II fragment in a DMPC lipid bilayer. The backbone conformations of DynA17 peptide11 and the ECL-II fragment were constructed according to the NMR results and minimized with the same procedure as in our previous reports.27,28 DynA17 and ECL-II were placed in the membrane at a distance where they cannot interact with each other. Their orientations in the membrane were set according to the NMR experimental results. That is, DynA17 and ECL-II were inserted into hydrated DMPC lipid bilayer with the helical region of DynA17 and the second α-helical region of ECL-II located near the membrane interface. The membrane builder module29 of CHARMM-GUI30 was used to build the initial conformation for the MD simulation with a concentration of 100 mM NaCl. The final model includes the DynA17 peptide and ECL-II fragment, 96 lipid and 4192 water molecules, 10 sodium and 9 chloride ions, for a total of 24774 atoms in the system. The dihedral angles of the backbone of helical regions of DynA17 and ECL-II were constrained and isobaric−isothermal (NPT) ensemble was carried out with 1 fs time steps for 30 ns using lab-level workstations. The details of the calculation procedure were the same as in our previous simulations.27,28,31

Figure 1. Effect of temperature variations of 31P NMR spectra of the Large Bicelle−ECL-II−DynA17 system with a molar ration of 200:1:1. Temperatures were changed as follows: 40 °C → 30 °C → 20 °C → 15 °C → 30 °C → 40 °C.

bicelles was confirmed at the temperature range of 15−40 °C, although magnetically aligned bicelles were estimated to be around 25% at 40 °C from the line-shape analysis. Figure 2a shows the 13C DD-MAS NMR spectrum of a small bicelle (q = 1) at 40 °C in comparison with that of a large bicelle (q = 3) (Figure 2b). The resolution of the 13C NMR spectrum of the carbonyl carbon nuclei for the small bicelle (q = 1) is much higher than for the large bicelle (q = 3). This can be attributed to the fact the mobility of the small bicelle (q = 1) is much faster than that of the large bicelle (q = 3). The

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RESULTS AND DISCUSSION P and 13C NMR Spectra of Bicelles Containing ECL-II and DynA17. Figure 1 shows the temperature variation of the 31 P DD-static NMR spectra of bicelles (DMPC:DHPC = 3:1) containing ECL-II and DynA17 with a composition of DMPCDHPC:ECL-II:DynA17 = 200:1:1. At the temperature above the phase transition temperature (Tc), spectra show that the bicelle exibits a lamellar phase. When the temperature decreased to 20 °C, anisotropic signals were largely decreased and consequently the isotropic signal was increased. When the temperature decreased to 15 °C, only an isotropic peak was observed. This result indicates that the bicelle phase changes the lamellar to an isotropic phase, consistent with a previously reported property of bicelles.32 When the temperature was increased to 40 °C, the perpendicular edge was increased, leading to observation of partial magnetic alignment, which is a well-known feature of large bicelles.33 Thus, the formation of 31

Figure 2. 13C DD-MAS NMR spectra of the carbonyl carbons of (a) a small bicelle (DMPC:DHPC = 1:1, q = 1) and (b) a large bicelle (DMPC:DHPC = 3:1, q = 3). 9606



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The isotropic chemical shift values showed that the secondary structure around Gly3 exhibits an α-helix structure (Table 1). On the other hand, the 13C NMR signal of [1-13C]Val12 moved significantly to the lower field with the addition of DynA17 to the ECL-II-bicelle system (Figure 4). This result indicates that

timescale of the motional frequency for a small bicelle is larger than 5 kHz because single 31P NMR spectrum (data not shown) was observed as a result of averaging out of 5 kHz anisotropy. 13C NMR peaks at 174.2 ppm for the large bicelle (174.1 ppm for the small bicelle) and 173.7 ppm for the large bicelle (173.7 and 173.6 ppm for the small bicelle) were assigned to carbonyl carbons of DHPC and DMPC, respectively. 13 C NMR Spectra of ECL-II Bound to Bicelles in the Absence and Presence of DynA17. Figure 3 shows the 13C

Figure 4. Effect of DynA17 concentration dependence on 13C DDMAS NMR spectra of the [1-13C]Val12-ECL-II system. Bicelle:ECLII:DynA17 = (a) 200:1:0, (b) 200:1:1, (c) 200:1:2, and (d) 200:1:5 for small bicelles. Marks ▼ indicate the signal positions of [1-13C]Val12ECL-II.

DynA17 perturbs the chemical shift value at the position of Val12. Since a single 13C NMR peak appeared in the [1-13C]Val12-ECL-II spectra, the exchange rate between ECL-II and the ECL-II−DynA17 complex is very rapid, resulting in the two signals averaging into a signal. It is also stressed that the structure of ECL-II at Val12 is perturbed by DynA17 and consequently forms a more rigid α-helix according to the conformation dependent [1-13C]Val chemical shift value.34−36 It is of interest to note that the chemical shift changes of ECL-II at the position of Ala15 in the large bicelle (q = 3) are larger than those in the small bicelle (q = 1) (Table 1). This result indicates that the interaction of DynA17 with ECL-II in the large bicelle is stronger than in the small bicelle. Since bilayer area of large bicelles is larger than that of small bicelles,

Figure 3. Effect of DynA17 concentration on 13C DD-MAS NMR spectra of the [1-13C]Gly3-ECL-II system. Bicelle:ECL-II:DynA17 = (a) 200:1:0, (b) 200:1:1, and (c) 200:1:5 for small bicelles and bilelle:ECL-II:DynA17 = (d) 200:1:0, (e) 200:1:1, and (f) 200:1:5 for large bicelles. Marks ▼ indicate the signal positions of [1-13C]Gly3ECL-II.

DD-MAS NMR spectra of [1-13C]Gly3-ECL-II at a variety of DynA17 concentrations. In the absence of DynA17, the 13C NMR signal of [1-13C]Gly3 appeared at 172.2 and 172.0 ppm for the bicelles of q = 3 and 1, respectively (Figure 3, panels a and d). These signals did not move significantly with the addition of DynA17 to the ECL-II-small and large bicelle systems (Figure 3, panels b and c, and Figure 3, panels e and f).

Table 1. 13C Chemical Shift Values δ(ppm) of the ECL-II-bicelle System at a Variety of DynA17 Concentrations amino acids 13

[1- C]Gly3 [1-13C]Val6 [1-13C]Val10 [1-13C]Val12 [1-13C]Ile13 [3-13C]Ala15 [1-13C]Leu17 [1-13C]Phe19

200:1:0a

200:1:1a

200:1:2a

± ± ± ± ± ± ± ± ± ± ±

172.0 ± 0.02 172.3 ± 0.05c −e 174.2 ± 0.04 173.6 ± 0.15c 174.9 ± 0.03 175.0 ± 0.05 16.3 ± 0.02 16.6 ± 0.10c 173.3 ± 0.03 171.6 ± 0.05c

172.1 ± 0.02 172.2 ± 0.05c −e 174.4 ± 0.04 −c,e 175.0 ± 0.03 175.0 ± 0.05 16.3 ± 0.02 16.1 ± 0.10c 173.3 ± 0.03 171.7 ± 0.05c

172.0 172.2 173.6 173.8 173.4 174.5 174.7 16.5 16.7 173.2 171.7

0.02 0.05c 0.02 0.04 0.15c 0.03 0.05 0.02 0.10c 0.03 0.05c

200:1:5a 172.2 172.1 173.7 174.5 −c,e 175.4 175.2 16.3 16.0 173.3 171.6

Δδb

structuresd

± ± ± ±

0.02 0.05c 0.02 0.04

0.2 0.1 0.1 0.7

± ± ± ±

0.04 0.10c 0.04 0.08

α-helix α-helix random coil α-helix

± ± ± ± ± ±

0.03 0.05 0.02 0.10c 0.03 0.05c

0.9 0.5 0.2 0.7 0.1 0.1

± ± ± ± ± ±

0.06 0.10 0.04 0.20c 0.06 0.10c

α-helix α-helix α-helix α-helix random coil random coil

Composition of bicelles: bicelle:ECL-II:DynA17. bChemical shift deference: Δδ = |δ(200:1:5) − δ(200:1:0)|. cChemical shift values in a large bicelle. dEstimated secondary structure by refs 34−36. eNot measured.

a

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ECL-II embedded in large bicelles may differ from that in small bicelles, which causes the difference of affinity to DynA17. Namely ECL-II embedded in large bicelles might have higher affinity than that in small bicelles. These chemical shift values are summarized in Table 1, together with the values of [1-13C]Gly3, Val6, Val10, Val12, Ile13, Leu17, and Phe19 and [3-13C]Ala15 of ECL-II in a variety of DynA17 concentrations. The conformation-dependent 13C chemical shift values34−36 indicate that the N-terminal region and Val10−Ala15 of ECL-II form α-helix structures. Chemical shift perturbations were significant in the range from Val10 to Ala15, while they were not significant at Gly3, Val6, Leu17, and Phe19 of ECL-II (Figure 5). These results indicate that DynA17 may bind at the region from Val10 and Ala15 of ECL-II, which is the helical region of ECL-II embedded in the lipid bilayers.

Figure 6. Representative time-dependent frequency reduction of DMPC-immobilized QCM responding to the addition of (a) 40, (b) 80, (c) 120, and (d) 160 μM of DynA17.

[complex]t = [complex]max

K a[DynA17] K a[DynA17] + 1

{1 − exp[−(1/τ ) ·t ]}

(3)

where τ is a decay time constant as defined by 1/τ = ka[DynA17] + kd

(4)

In the QCM studies, the amount of DynA17 binding is monitored by changes in frequency (ΔF). Therefore, eq 3 is described by

Figure 5. Plot of chemical shift difference (perturbation) of the 13C chemical shift values in the presence of DynA17 (Bicelle:ECLII:DynA17 = 200:1:5) from those in the absence of DynA17 (Bicelle:ECL-II:DynA17 = 200:1:0). Gray bars indicate the values obtained using small bicelles and white bars indicate the values from large bicelles.

ΔFt = ΔFmax

K a[DynA17] {1 − exp[−(1/τ ) ·t ]} K a[DynA17] + 1

(5)

The linear regression analysis of the relationship between 1/τ and DynA17 concentration is shown in Figure 7. In accordance

Kinetics Analysis of Interactions of DynA17 with DMPC and ECL-II. In order to monitor the binding of DynA17 to DMPC and ECL-II, DMPC monolayers, or ECL-II reconstituted into DMPC were immobilized on an noctadecanethiol modified QCM electrode surface. Figure 6 shows the frequency changes observed following the addition of DynA17 at various concentrations into DMPC-immobilized QCM. These results indicate that the amount and rate of DynA17 binding to DMPC increased with increasing concentrations of DynA17. The kinetics of the interaction between DynA17 and DMPC was analyzed using the frequency decay data at various concentrations of DynA17. The relationship of the interaction between DynA17 and DMPC can be described as follows, Ka

DMPC + DynA17 ↔ complex

(2)

where Ka is the association constant and given by ka/kd using the association (ka) and dissociation (kd) rate constants. In the QCM studies, the amount of DynA17 binding is monitored by changes in frequency (ΔF). Therefore, the time course of the complex formed by DynA17 binding to DMPC is given by

Figure 7. Linear regression analysis of the relationship between τ−1 and DynA17 concentration in DynA17 binding to DMPCimmobilized QCM. The decay time constant (τ) was calculated using eq 3. 9608



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with eq 4, ka and kd were determined from the slope and intercept of the regression line to be 44.3 s−1 M−1 and 3.4 × 102 s−1, respectively. The association constant Ka was calculated to be 1.3 × 104 M−1 from ka/kd. This affinity indicates that DynA17 exhibits only a weak interaction with DMPC membranes. Affinity Analysis of the Interaction of DynA17 with ECL-II. In order to analyze the interaction between DynA17 and ECL-II reconstituted into DMPC, a variety of molar mixture ratios of ECL-II to DMPC (1:200, 1:100, and 1:50) were immobilized on a QCM electrode surface. Linear regression analysis of the relationship between τ−1 and DynA17 concentration for a variety of lipid compositions of DMPC and ECL-II were performed as shown in Figure 8.

equilibrium (ΔF∞) and the concentration of DynA17. In eq 5, ΔF∞ is described by ΔF∞ = ΔFmax

K a[DynA17] K a[DynA17] + 1

(6)

where the ΔFmax is the maximum frequency change when DynA17 is bound to all of the binding sites on QCM. When DynA17 coincidentally interacts with DMPC and ECL-II, the relationship can be described as follows: ΔF∞ = ΔFmax(ECL ‐ II)

K a(ECL ‐ II)[DynA17] K a(ECL ‐ II)[DynA17] + 1

+ ΔFmax(DMPC)

K a(DMPC)[DynA17] K a(DMPC)[DynA17] + 1

(7)

where Ka(ECL‑II) and Ka(DMPC) are the association constants for DynA17 interactions with ECL-II and DMPC, respectively. The relationship between ΔF∞ and the concentration of DynA17 ([DynA17]), where ECL-II is reconstituted into 50fold DMPC, is represented in Figure 9. Using 1.3 × 104 M−1 as

Figure 8. Linear regression analysis of the relationship between τ−1 and DynA17 concentration. Molar ratio lipid compositions of DMPC and ECL-II are (a) 200:1 plotted as ◇, (b) 100:1 plotted as □, and (c) 50:1 plotted as ▲. Figure 9. Saturability of the interaction between DynA17 and ECL-II reconstituted in DMPC. The lipid composition molar ratio of DMPC and ECL-II is 50:1. The fitted curve was evaluated from eq 7.

Kinetics parameters could be calculated from the eqs 4 and 5. The obtained ka, kd, and Ka values are summarized in Table 2.

the Ka(DMPC) value (summarized in Table 2), the Ka(ECL‑II), ΔFmax(ECL‑II), and ΔFmax(DMPC) values are estimated to be 9.4 × 105 M−1, 28.4 Hz, and 49.4 Hz, respectively, from eq 7. The estimated Ka(ECL‑II) value was 72 times as large as Ka(DMPC). Therefore, DynA17 appears to have specifically interacted with ECL-II. The total of the maximum frequency change (ΔFmax(ECL‑II) + ΔFmax(DMPC)) observed was 77.8 Hz. The ΔFmax value was estimated to be 131.8 Hz for DMPC alone and is larger than the value of mixed lipid compositions (ΔFmax(ECL‑II) + ΔFmax(DMPC)). This indicates that the ECL-II is dominated on DMPC, and DynA17 binding sites are decreased on the surface. MD Simulation of Interaction between DynA17 and ECL-II. MD simulation of the DynA17 and ECL-II fragment in a DMPC bilayer was performed, and the interactions between these two molecules were investigated. Figure 10a shows the time course of the interaction energy between the DynA17 peptide and the ECL-II fragment in a DMPC bilayer. The interaction energy was found to begin increasing from around 9 ns and continues its interaction until the end of the simulation. That is, DynA17 and the ECL-II fragment begin to interact

Table 2. Estimated Association (ka), and Dissociation (kd) Rate Constants, and Association Constant (Ka) for Various Lipid Composition Ratios of DMPC and ECL-II lipid composition (DMPC:ECL-II)

ka (s−1 M−1)

1:0 200:1 100:1 50:1

44.3 66.5 1.5 × 102 4.0 × 102

kd (s−1) 3.3 4.1 1.4 7.2

× × × ×

10−3 10−3 10−3 10−4

Ka (M−1) 1.3 1.6 1.1 5.6

× × × ×

104 104 105 105

The ka values decreased and kd increased as the ECL-II content ratio increased. As a consequence, Ka values increased with increasing ECL-II content ratios in DMPC. This indicates two conclusions: the DynA17 has high affinity for ECL-II, and DynA17 binds to ECL-II as well as to DMPC in this experimental system. The Ka value of the interaction between DynA17 and ECL-II embedded into DMPC lipids was altered according to the composition of DMPC and ECL-II. In order to estimate the Ka value between DynA17 and ECL-II, the association constant (Ka) is calculated using the degree of frequency change at 9609



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Figure 11. Distribution of the interaction energy between the ECL-II fragment and DynA17 peptide amino acid residues are shown in (a). Interaction energies were averaged over the last 10 ns of the simulation. The horizontal bars represent the average values. Snapshots of the interacting residues between the ECL-II fragment and the DynA17 peptide at 30 and 29 ns are shown in (b) and (c), respectively. The interacting residues of Lys11, Lys13, and Trp14 of DynA17 and Val10, Ile13, Glu14, and Gln18 amino acid residues of ECL-II are shown as “Licorice” representations.

Figure 10. (a) MD analysis of the time course of the interaction energy between the ECL-II fragment and the DynA17 peptide. Snapshots of DynA17 peptide (blue) and ECL-II fragment (green) in the DMPC membrane bilayer at the (b) start and (c) end of 30 ns MD simulation. Lipid and water molecules are shown in gray and red, respectively. For simplicity, sodium and chloride ions are not shown.

with each other after 9 ns in this simulation. Snapshots of the molecules at the beginning and end of the simulation are shown in Figure 10, panels b and c, respectively. It can be seen that the molecules are located separately at the beginning of the simulation and approach and interact with each other at the end of the simulation. Figure 10c shows that the C-terminus of DynA17 peptide interacts with the N-terminal α-helical region of the ECL-II fragment. This interaction occurs in the aqueous phase, whereas the other regions of the molecules remain in the bilayer. The interaction may fluctuate according to the flexible movement of the interacting section in the water environment. The interaction between these two molecules was analyzed in more detail, and the results are shown in Figure 11a. This figure shows the interaction energy between each residue of the ECLII fragment and the DynA17 peptide. The interaction energies are shown as averaged values over the last 10 ns of the simulation. The results show that the DynA17 peptide interacts with the amino acid residues of the regions from Val10 to Glu14 of the ECL-II fragment. This result is in good agreement with the experimental results of chemical shift perturbation. However, the time course of the interaction energy in Figure 10a indicates the possibility of the existence of two interaction modes. That is, the time regions with strong interactions with the same orientation appeared at approximately 16 and 29 ns, and the interactions continue for 1 ns in both cases. The other time segments of the simulation show interaction energies of greater than −50 kcal/mol. To investigate the interaction behavior in more detail, a snapshot of the typical orientation of DynA17 and the ECL-II fragment, extracted during a weak interaction region at 30 ns, is shown in Figure 11b. It can be seen that the Trp14 of DynA17 locates close to the Val10 and

Ile13 residues of the ECL-II fragment, which indicates that the molecules interact with each other throughout these residues. As the methyl protons of Val10 and Ile13 of ECL-II locate above the aromatic ring of Trp14, they should interact with a CH/π interaction. Similar interactions have been found in many biological systems and reported in many recent studies.37 Conversely, a snapshot of a typical orientation of DynA17 and ECL-II fragments during a strong interaction at 29 ns is shown in Figure 11c. In this case, the Lys11 of DynA17 locates close to the Gln18 residues of the ECL-II fragment, and Lys13 of DynA17 locate close to the Glu14 residue of the ECL-II fragment. As these paired residues are charged with opposite polarity, electrostatic interactions should occur between these residue pairs. These electrostatic interactions would produce a strong interaction with values of approximately −150 kcal/mol. However, the duration of these interactions cannot be longlived and will fluctuate in the aqueous environments. Binding Site and Structural Alternation of ECL-II in the Presence of DynA17. Figure 5 is a plot of the chemical shift perturbations at the various amino acid positions of ECLII in the presence of DynA17. This result clearly indicates that DynA17 strongly interacts with the amino acid location between Val10 and Ala15 of ECL-II. In our previous work, the membrane-bound structure of DynA17 was determined, and it was found that the N-terminal α-helix is inserted into the membrane with the α-helical axis tilting 21° with respect to the bilayer normal.11 The present experiments revealed that ECL-II also exhibits an α-helix structure in the region from Val10 to Ala15. Although the helical region of DynA17 was predicted to 9610



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Present Address

interact with that of ECL-II, MD simulation results showed that the region of Val10 to Glu14 in the helical region of ECL-II interacts with the nonhelical C-terminal region of DynA17 through hydrophobic interactions between Trp14 in DynA17 and Val10 and Ile13 in ECL-II (Figure 11b) together with short-lived electrostatic interaction between Glu14 and Gln18 of ECL-II, and Lys13 and Lys11 of DynA17, as shown in Figure 11c. These specific interactions of the side chains may cause the chemical shift perturbation due to conformational changes in the ECL-II α-helix in the range from Val10 to Ala15. Although Val12 shows large chemical shift displacements by interacting with DynA17, a single NMR peak was detected (Figure 4). This result indicates that DynA17 is bound to the membrane, and molecules bound to ECL-II underwent rapid exchange. On the other hand, the exchange rate between DynA17 bound to the membrane itself and the monomer state of DynA17, is significantly slower (Figure 12). It was observed that DynA17

†

Product Development Center, Japan Aviation Electronics Industry, Ltd., 3-1-1 Musashino, Akishima, Tokyo 196-8555, Japan. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for Scientific Research on Priority Area (24121709) and Innovative Area (26102545 and 26104513), and a Grant-in-aid for Scientific Research (C) (24570127) from the Ministry of Culture, Sports, Science and Technology of Japan and the NAITO FOUNDATION. The authors wish to thank the Research Center for Computational Science, Okazaki, Japan, for the use of their computer to perform part of the calculations present here.

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Figure 12. Schematic representation of the process of DynA17 binding to ECL-II.

first interacts with the membrane to form an N-terminal α-helix consistent with the membrane catalytic model.38,39 Subsequently, DynA17 interacts with ECL-II more efficiently because DynA17 readily diffuses on the surface of the membrane. It is reported that the active site of the κ-opioid receptor is located in the core of the receptor.38 In this case, the binding site for DynA17 on ECL-II is considered to be the address site. Consequently, it becomes efficient for Tyr in the message site at the N-terminal end to bind to the active site of the receptor.

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CONCLUSIONS Here, it has been clearly demonstrated using 13C NMR, QCM, and MD simulation that ECL-II exhibits strong affinity toward the opioid peptide DynA17 in membrane environments. The structure of ECL-II bound to lipid bilayers was determined to be an α-helix, and the Val10-Ala15 region of this extracellular helix interacts with the nonhelical C-terminal region of DynA17 through mainly hydrophobic interactions. This specific interaction is dynamic in nature because the hydrophobic and short-lived electrostatic interactions are alternatively changed as well as the rapid exchange between ECL-II-DynA17 and ECLII in the membrane-bound state. The present description of the nature of the ECL-II-DynA17 complex provides insight into the mechanism of receptor ligand interaction.

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REFERENCES

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