Exploring Deltorphin II Binding to the Third Extracellular Loop of the δ-Opioid Receptor

The third extracellular loop of the human δ-opioid receptor (hDOR) is known to play an important role in the binding of δ-selective ligands. In particular, mutation of three amino acids (Trp284, Val296, and Val297) to alanine significantly diminished δ-opioid receptor affinity for δ-selective ligands. To assess the changes in conformation accompanying binding of the endogenous opioid peptide deltorphin II to the δ-opioid receptor at both the receptor and ligand levels as well as to determine points of contact between the two, an in-depth spectroscopic study that addressed these points was initiated. Fragments of the δ-opioid receptor of variable length and containing residues in the third extracellular loop were synthesized and studied by NMR and CD spectroscopy in a membrane-mimetic milieu. The receptor peptides examined included hDOR-(279–299), hDOR-(283–299), hDOR-(281–297), and hDOR-(283–297). A helical conformation was observed for the longest receptor fragment between Val283 and Arg291, whereas a nascent helix occurred in a similar region for hDOR-(281–297). Further removal of N-terminal residues Val281 and Ile282 abolished helical conformation completely. Binding of the δ-selective ligand deltorphin II to hDOR-(279–299) destabilized the helix at the receptor peptide N terminus. Dramatic changes in the α-proton chemical shifts for Trp284 and Leu286 in hDOR-(279–299) also accompanied this loss of helical conformation. Large upfield displacement of α-proton chemical shifts was observed for Leu295, Val296, and Val297 in hDOR-(279–299) following its interaction with deltorphin II, thus identifying a gain in β-conformation at the receptor peptide C terminus. Similar changes did not occur for the shorter peptide hDOR(281–297). A hypothesis describing the conformational events accompanying selective deltorphin II binding to the δ-opioid receptor is presented.

Opioid receptors are widely recognized for their role in mediating pain. Recent cloning of the cDNAs encoding the three opioid receptor subtypes (, , and ␦) defined them as belonging to the superfamiliy of G protein-coupled receptors. Upon comparison of the different opioid receptor subtypes, considerable amino acid identity has been found in the transmembrane and intracellular regions, with a 60% receptor homology overall (1)(2)(3)(4). Greater sequence diversity occurs in the extracellular loops and toward the N and C termini.
The ␦-opioid receptor represents a particularly attractive target for the development of pain therapeutics because of its known ability to mediate analgesia without inducing opiate physical dependence. Furthermore, ␦-selective drugs may possess potential clinical benefits over those currently targeted toward the -opioid receptor (5)(6)(7)(8). These advantages include greater relief of neuropathic pain and reduced respiratory depression and constipation as well as a minimal potential for the development of physical dependence (9).
Deltorphin II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH 2 ), originally isolated from frog skin, represents the most ␦-selective ligand among the endogenous opioid peptides discovered so far (10). Structure-activity relationship studies centered on the deltorphins have revealed a number of key elements within the peptide sequence that are important for affinity and or selectivity (9). A negative charge on the Glu 4 side chain is critical for ␦-receptor selectivity, but not for receptor affinity (11,12), whereas the presence of hydrophobic residues at Val 6 and Val 7 is required for both high affinity binding and ␦-receptor selectivity (13)(14)(15). Removal of the Tyr 1 hydroxyl group results in loss of ␦-receptor affinity (16). High ␦-receptor selectivity may also be attributed to a compact peptide conformation, which is postulated as preferred by the ␦-opioid receptor (17).
Evidence gathered from both site-directed mutagenesis and chimeric receptor studies as well as from subsequent molecular modeling studies have pointed to a number of residues located in the transmembrane helices and extracellular loops of human ␦-opioid receptor (hDOR) 1 that are potentially involved in the binding of both ␦-selective agonists and antagonists (18 -21). In particular, simultaneous mutation of three residues (Trp 284 , Val 296 , and Val 297 ) to alanine in the third extracellular loop of hDOR significantly alters the normal binding of the ␦-selective ligands SNC-80, Tyr-D-Pen-Gly-Phe-D-Pen-OH (DPDPE), deltorphin II, and naltrindole (19). Others studies have suggested that Leu 300 and Arg 291 as well as the hydrophobic nature of hDOR-(295-300) are important for ␦-selective binding (21).
In this study, we have undertaken the structural characterization of the third extracellular loop (ECLIII) of hDOR both alone in solution and in a complex with deltorphin II. The goal was to investigate changes occurring at both the receptor peptide and ligand levels following formation of the complex that may explain events following deltorphin II binding to hDOR. Four peptides of different length containing the ECLIII and some of the transmembrane amino acids of hDOR were syn-thesized. The conformations of these peptides in 88% hexafluoroisopropyl alcohol (HFIP) and 12% H 2 O were assessed using CD and NMR spectroscopy. The results suggest that the interaction of deltorphin II with the hDOR ECLIII generally destabilizes a helix at the N terminus of the extracellular loop and promotes a ␤-structure at its C terminus.

EXPERIMENTAL PROCEDURES
Materials-The chemical structures of the opioid receptor ligands used in this study are shown in Fig. 1. Deltorphin II and dynorphin A-(1-13) were obtained from Bachem California. High purity endomorphin-1 and -2 were purchased from Bachem Bioscience Inc. U-50488 and sodium P i were from Sigma. Trifluoroethanol (TFE) and HFIP were obtained from Aldrich. SDS was purchased from Bio-Rad.
Peptide Synthesis-The linear hDOR ECLIII peptides hDOR-(283-297), hDOR-(283-299), hDOR-(281-297), and hDOR-(279 -299) (Fig. 2) were synthesized on solid support using a Symphony multiple peptide synthesizer and standard Fmoc chemistry procedures with HATU as coupling reagent. The polymeric starting material was CLEAR amide resin and TentaGel R RAM (Peptides International Inc.) with 0.4 and 0.22 mmol/g resin loading capacity, respectively. The crude peptides were cleaved from the resin by trifluoroacetic acid/triisopropylsilane/ thioanisole/H 2 O (94:2:2:2) and then isolated by ether precipitation. Peptides were subsequently purified to homogeneity by preparative chromatography on a Hitachi HPLC instrument equipped with a Jupiter RP-4 column (250 ϫ 22 mm). The synthesized products were obtained in high yield with purity Ն 96% as determined by analytical high performance liquid chromatography on a Jupiter RP-18 column and a Vydac 218TP54 column and by liquid chromatography-mass spectrometry. Electrospray mass spectrometry using a Micromass LCT mass spectrometer provided the correct molecular weight for each peptide. All peptides were amidated at the C terminus.
Sample Preparation-Samples for CD spectroscopic studies were prepared by dissolving a sufficient amount of peptide in 500 l of one of three solvent systems (440 l of TFE and 60 l of H 2 O, 440 l of HFIP and 60 l of H 2 O, and 200 mM SDS in 50 mM sodium P i (pH 6.0)) to provide a final peptide concentration of 2 mM, unless otherwise stated. NMR samples contained 2 mM peptide in 440 l of HFIP and 60 l of H 2 O. All non-aqueous reagents used for NMR experiments were fully deuterated. The solvents systems were chosen to provide a membranemimetic environment and to maximize peptide solubility.
For studies aimed at examining the interaction between opioid ligands and various hDOR ECLIII peptides, a 2 mM sample of the chosen receptor peptide was first prepared in HFIP/H 2 O as described above. The ligand was then added to a peptide concentration of 2 mM.
CD Spectroscopy-CD spectra were recorded on a Jasco J710 spectropolarimeter at room temperature. Ten scans were collected for each sample over a wavelength range of 180 -260 nm under the following conditions: 0.2-nm resolution, 1.0-nm bandwidth, 100-nm/min scan speed, and 0.25-s response time. The CD cell path length employed for all experiments was 0.01 cm. The collected spectra were improved through background subtraction and smoothing and then converted to units of molar ellipticity/residue (degrees cm 2 dmol Ϫ1 ). Estimates of the helical fraction of the peptide were calculated by the method of Green-field and Fasman (22) using a value of [] at 222 nm.
NMR Spectroscopy-All NMR spectra were acquired on a Bruker Avance-600 spectrometer at 300 K. 3,3,3-Trimethylsilyl propionate was used as an internal reference (0.0 ppm). 1 H signals for each peptide were assigned from analysis of the cross-peaks in total correlation spectroscopy (TOCSY) spectra acquired with a 50-ms mixing time and from the sequential connectivities determined from nuclear Overhauser effect (NOE) correlation spectroscopy spectra obtained with a mixing time of 200 ms. Two-dimensional TOCSY and NOE correlation spectroscopy experiments were carried out in the phase-sensitive mode using the States-time-proportional phase incrementation (TPPI) method. In cases where TFE/H 2 O and HFIP/H 2 O solvent mixtures were employed, the solvent signals appeared as two broad peaks, one originating from the water protons and the other from the residual alcohol OH. Chemical exchange between the two labile proton sites resulted in similar solvent peak intensities. The best overall solvent suppression was thus achieved by presaturating one of the solvent peaks for a period of 1 s prior to the first pulse of each free induction decay acquisition.
The amide proton exchange rates in deltorphin II were measured by first incubating a 2 mM solution of peptide in 440 l of HFIP and 60 l of D 2 O for 24 h and then lyophilizing the sample. The lyophilized sample was resuspended in a mixture of 440 l of HFIP and 60 l of H 2 O, and two-dimensional TOCSY spectra were acquired at 15-min intervals immediately following dissolution. After 3 h of incubation, hDOR-(279 -299) was added to the HFIP/H 2 O sample at 2 mM, and spectral monitoring was continued.
A second amide exchange rate experiment involved first incubating the receptor peptide hDOR-(281-297) at 2 mM in HFIP/H 2 O and then lyophilizing the sample. Samples were dissolved in 440 l of HFIP and 60 l of D 2 O. Disappearance of the amide proton signals was monitored through acquisition of two-dimensional TOCSY spectra every 15 min following sample preparation. The experiment was repeated with deltorphin II also contained in the sample at 2 mM.

CD Spectroscopy
Optimization of Organic Solvent Conditions-A general study was performed in which the percentage of organic solvent (TFE or HFIP) in the alcohol/water solvent systems was varied, and the solubility of the hDOR ECLIII peptides was monitored.

FIG. 2. Linear hDOR ECLIII peptides examined in this study.
All peptides were synthesized on solid support using standard Fmoc chemistry procedures with HATU as coupling reagent. Peptides were subsequently purified to homogeneity by preparative chromatography. The final purities of all peptides were Ͼ95%. Fluorinated alcohol/water solvents systems have been widely employed for examining peptide conformations in a membrane-like environment. In addition, TFE and HFIP are structure-promoting solvents that are particularly effective in stabilizing helical conformations without inducing intermolecular aggregation (23)(24)(25).
Optimal solubility conditions were noted when the fraction of organic solvent reached 88%. Sample stability over time was marginally better when HFIP was employed as the organic solvent. To ensure that the conformation of the hDOR ECLIII peptides were not affected by a change in peptide concentration, CD spectra were obtained for the longest peptide, hDOR-(279 -299), in 440 l of HFIP and 60 l of H 2 O using four different peptide concentrations ranging from 2 to 0.1 mM. The CD spectra did not change over the entire concentration range studied. Therefore, the peptide did not aggregate at 2 mM. These controls were repeated for the remaining peptides, leading to the same conclusions.
Conformational Analyses-To assess the overall conformational preference of the hDOR ECLIII peptides, CD spectra were first acquired for the longest peptide, hDOR-(279 -299), in a number of solvent systems. As shown in Fig. 3, the CD spectrum of this peptide in HFIP/H 2 O contained a well resolved shoulder near 222 nm, a negative peak at 208 nm, and a positive peak at 192 nm. These spectral properties are consistent with a partial helical peptide. Almost an identical spectrum was observed when the peptide was dissolved in TFE/H 2 O (Fig.  3). However, the percentage helicity marked by the absorbance at 222 nm was more pronounced in this case. In SDS, the shape of the CD curve was typical of a peptide with ␤-sheet structure and contained a broad band centered at 216 nm (Fig. 3). The ␤-conformation likely reflects the presence of an aggregated peptide system in the detergent environment. Indeed, support for this statement stems from the observation of a white precipitate in the micellar sample.
When CD spectra were acquired for the remaining hDOR peptides in HFIP/H 2 O and compared with that obtained for hDOR-(279 -299), a number of similarities and differences were noted (Fig. 4). The spectrum for the medium length peptide hDOR-(281-297) displayed a partial ␣-helical conformation as judged by the observation of two negative peaks at 222 and 202 nm and a positive peak at 190 nm (Fig. 4). The left shift of the negative band at 202 nm indicated a destabilization of the helix compared with that associated with hDOR-(279 -299). Deletion of the N-terminal Ile 279 -Phe 280 motif and the C-terminal Ala 298 -Ala 299 motif in hDOR-(279 -299) thus had the effect of moderately destabilizing the secondary structure of the peptide. CD spectra acquired for the remaining medium length peptide, hDOR-(283-299), and smallest analog, hDOR-(283-297), suggested that less helix stability was associated with these two molecules. This result implies that further removal of N-terminal residues Val 281 and Ile 282 abolishes the helical conformation. Perhaps there is no longer a critical number of amino acids available for establishment of two helical turns along the peptide backbone. However, some conformational preference for the pair was evident from the shoulder at 222 nm and the positive peak at 190 nm in their associated CD spectra (Fig. 4).
␦-Opioid Receptor Peptide-Ligand Complexes-Changes in the CD spectrum of hDOR-(279 -299) in HFIP/H 2 O produced by adding opioid receptor ligands ( Fig. 1) were investigated. The CD spectrum acquired for hDOR-(279 -299) in the presence of deltorphin II exhibited a reduced negative ellipticity at 222 nm and a shift in the absorption maximum from 208 to 205 nm compared with that obtained for the receptor peptide alone (Fig. 5a). Interestingly, adding endomorphin-1 and -2 (endogenous ligands of the -opioid receptor) to a sample of hDOR-(279 -299) also resulted in reduced negative ellipticity at 222 nm (Fig. 5, b and c, respectively). Addition of a -selective peptide agonist, dynorphin A-(1-13), to a hDOR-(279 -299) sample actually increased very marginally the negative absorbance at 222 nm (Fig. 5d), suggesting that helix stabilization occurred. No change was observed in the hDOR-(279 -299) CD spectrum when a small molecule -selective agonist (U-50488) was added.
Addition of deltorphin II to samples of the other three hDOR ECLIII peptides in general did not change their CD spectra (data not shown). Only a small decrease was observed in the negative ellipticity of hDOR-(281-297) at 202 and 222 nm when deltorphin II was added, again reflecting destabilization of the helical conformation.

NMR Spectroscopy
More detailed conformational properties of the hDOR ECLIII peptides were examined by NMR spectroscopy. Initially, a search for secondary structure was performed using the chem- ical shift analysis method developed by Wishart et al. (26). As stipulated by this method, observed pronounced upfield ␣-H chemical shift deviations (Ͼ0.1 ppm) from random coil values within a stretch of four or more residues signal the presence of a helical structure. Conversely, a stretch of three or more pronounced ␣-H downfield resonance shifts defines a region of ␤-structure. Analysis of the ␣-H chemical shifts for the longest loop peptide, hDOR-(279 -299), indicated that there is a helix in the Val 283 -Arg 291 region (Fig. 6a). No such feature was observed in any of the other three peptides according to the chemical shift data alone (Fig. 6, b-d). However, a short density of upfield-shifted ␣-proton frequencies (Ͼ0.1 ppm) was noted in the Asp 289 -Arg 291 region in the case of hDOR-(281-297) (Fig. 6b). It is likely that a nascent helix populates this region of the peptide because the CD spectrum acquired for the same sample supports the presence of a weak helical structure. The main difference between hDOR-(279 -299) and hDOR-(281-297) at the N-terminal end is the absence of two transmembrane domain amino acids (Ile 279 and Phe 280 ). Therefore, a Ile 279 -Phe 280 sequence may play an important role in stabilizing an N-terminal helix in the hDOR ECLIII.
An additional noteworthy observation was the ␤-conformation formed at the C terminus of hDOR-(281-297) and hDOR-(283-297) (Fig. 6, b and d). Supporting evidence for this assessment was obtained from the NOE results. Two i,iϩ2 NOEs including Asp 293 ␣-H to Leu 295 NH and Pro 294 ␣-H to Val 296 NH were observed only in the case of hDOR-(281-297) and hDOR-(283-297). These data suggest the presence of a C-terminal ␤-turn structure. Because the remaining two peptides, hDOR-(279 -299) and hDOR-(283-299), end with an Ala 298 -Ala 299 motif, it may be hypothesized that the presence of these two amino acids destabilizes the ␤-structure.
When the ␣-H chemical shift analysis was repeated for the longest peptide, hDOR-(279 -299), in the presence of deltorphin II, considerable changes were noted. According to the ␣-H chemical shift data, a ␤-structure formed at the C terminus between Asp 293 and Val 297 . Moreover, the binding of deltorphin II destabilized the helix originally present in hDOR-(279 -299) (Fig. 7a). Dramatic changes in the hDOR-(279 -299) ␣-H chemical shifts occurred for five receptor amino acids: Trp 284 , Leu 286 , Leu 295 , Val 296 , and Val 297 .
Addition of deltorphin II to a sample of hDOR-(281-297) also resulted in an overall destabilization of the helical conformation between Asp 288 and Arg 291 . This is indicated by the shift in ␣-H chemical shift indices to more positive values and above the Ϫ0.1-ppm helix cutoff for amino acids in this region (Fig.  7b). When deltorphin II was added to samples of hDOR-(283-299) and hDOR-(283-297), no changes in their respective ␣-H chemical shifts occurred; and hence, peptide secondary structure was not affected in either case (data not shown).
When the focus of NMR analysis was shifted from the receptor peptides to the ligand deltorphin II, some interesting observations were made. A sample of deltorphin II was first incubated in HFIP/D 2 O to allow an NH-to-ND exchange among the amide protons. The sample was then freeze-dried and resuspended in HFIP/H 2 O. The degree to which each amide proton was shielded from the aqueous solvent was determined by examining the rate of reappearance of the NH signals in TOCSY spectra over time. Within a short period of time (15 min) after dissolving the peptide, all the NH signals reappeared with reasonable intensity, except for the amide proton signal associated with Glu 4 . Absence of a Glu 4 NH signal was still apparent 3 h following dissolution of the peptide in the HFIP/H 2 O milieu (Fig. 8a). This result suggests that the amide proton of Glu 4 is engaged in a hydrogen bond or is otherwise inaccessible to the surrounding water molecules. When the receptor peptide hDOR-(279 -299) was added to the same sam-ple at the 3-h time point, the Glu 4 NH signal intensified to the same level as those of the other amide protons. Disruption of the shielding process around this amide proton therefore occurred as a result of a deltorphin II-receptor peptide interaction.
The complex formed between deltorphin II and the hDOR ECLIII surrogate was examined in more detail. hDOR-(281-297) was chosen over hDOR-(279 -299) for these experiments because it was easier to work with in terms of sample stability. Furthermore, hDOR-(281-297) exhibited similar changes in its CD spectrum following an encounter with deltorphin II compared with the longer hDOR ECLIII peptide. A sample of hDOR-(281-297) was first lyophilized from 440 l of HFIP and 60 l of H 2 O and then resuspended in HFIP/D 2 O at the same solvent ratio. After 1.5 h, all of the amide proton signals had disappeared, except for those belonging to Val 287 and Val 296 (Fig. 9). When the experiment was repeated with both hDOR-(281-297) and deltorphin II contained in the sample, a number of receptor peptide amide protons exhibited slow NH-to-ND exchange. In particular, the amide proton signals associated with Thr 285 , Leu 286 , Val 287 , Leu 295 , Val 296 , and Val 297 were still visible 1.5 h following sample preparation. In the case of deltorphin II, only the Gly 7 amide proton exhibited slow exchange (Fig. 9). DISCUSSION In this study, the conformations of four synthetic peptides representing the hDOR ECLIII were examined in solution both alone and in the presence of opioid receptor-selective ligands. Each receptor-mimetic peptide involved in this study contained the entire extracellular portion of the hDOR ECLIII and some of the hydrophobic transmembrane amino acids. The purpose of this study was first to determine the propensity of the hDOR ECLIII to form secondary structures and second to gain insight into the mechanism of complex formation between hDOR and deltorphin II. This work focused on three critical hDOR ECLIII amino acids (Trp 284 , Val 296 , and Val 297 ) as previously determined through molecular biology experiments (19).
The results presented here suggest that the longest hDOR ECLIII peptide, hDOR-(279 -299), adopts a well defined helical conformation within the N-terminal portion of its amino acid sequence. Elimination of the Ile 279 -Phe 280 motif from the N terminus of hDOR-(279 -299) destabilized the helical conformation. It is thus conceivable that an aromatic or hydrophobic interaction between the side chains of Phe 280 and Trp 284 is required for stabilizing the helix whose starting point is Val 283 according to ␣-proton chemical shift data (Fig. 6). Phe 280 and Trp 284 are separated by three amino acids; and hence, their respective side chains are appropriately positioned for interaction, assuming a helical conformation exists. A side chain hydrophobic interaction is more likely the case, however, because mutation of Phe 280 to alanine in hDOR does not appreciably affect the binding of ␦-opioid receptor ligands (19). Interestingly, complete abolishment of the helical conformation was evident following removal of four additional N-terminal amino acids in hDOR-(279 -299). This result suggests that the shortened peptide hDOR-(283-299) no longer has the critical number of amino acids required for promoting an N-terminal helical fold.
The interaction of deltorphin II with the longest hDOR peptide, hDOR-(279 -299), resulted in destabilization of the helix (Figs. 6 and 7a). Dramatic displacement of the ␣-H chemical shifts for Trp 284 and Leu 286 accompanied this change, indicating an interaction between the receptor loop peptide and deltorphin II in the vicinity of Trp 284 (Figs. 6 and 7a). It has been demonstrated through mutagenesis experiments that Trp 284 plays a major role in the hDOR-selective binding of opioid ligands (19). Previous structure-activity relationship investigations have also provided evidence indicating that the size and hydrophobic nature of Phe 3 in addition to the negative charge associated with Glu 4 of deltorphin II are critical determinants for hDOR binding. In particular, the acidic function associated with Glu 4 is considered a discriminating factor for ␦-opioid receptor selectivity (11,12,(27)(28)(29)(30)(31). Upon comparison of the amino acid sequences in the vicinity of the ECLIII for both the ␦and -opioid receptors, one notices two positively charged residues in the hDOR sequence (Arg 291 and Arg 292 ), whereas the -opioid receptor is void of any acidic residues in this region. An intensive site-directed mutagenesis study of hDOR also identified Arg 291 as an important residue for the binding of deltorphin II, suggesting that the Glu 4 side chain of deltorphin II may interact with Arg 291 of the receptor (19). The closest distance of approach between the side chains of Trp 284 and Arg 291 measured from a crude helical model of hDOR-(279 -299) is 4.7 Å (data not shown). It can be hypothesized then that deltorphin II Phe 3 and Glu 4 interact with hDOR Trp 284 and Arg 291 , respectively. The loss of a helical conformation may arise from the disruption of helix-stabilizing forces, including possibly interruption of a Phe 280 -Trp 284 interaction, resulting from the ligand-receptor interaction. Further support for this hypothesis stems from the results of amide proton exchange experiments. When a ligand-receptor interaction was allowed to occur, the amide protons of Thr 285 , Leu 286 , and Val 287 exhibited a slow exchange with the surrounding aqueous milieu. By contrast, these same protons exchanged rather quickly with deuterium when deltorphin II was absent. These data suggest that the amide protons close to Trp 284 become more shielded from the solvent in the presence of deltorphin II presumably due to an interaction between deltorphin II and hDOR-(281-297) around Trp 284 of the receptor peptide. It should be pointed out that the amide protons of the receptor peptide alone should also be well shielded form the solvent and exhibit a slow NHto-ND exchange if a very stable helix is present. The fact that fast exchange was observed for hDOR-(281-297) amide protons in this case reflects the nascent helix determined for this particular receptor peptide.
From the ligand point of view, some changes were noted around Glu 4 following a deltorphin II-hDOR-(281-297) interaction. Weak Glu 4 amide proton signal intensity was consistently found in TOCSY spectra acquired over time for deltorphin II in solution (Fig. 8a). These data reflect a slow NDto-NH exchange of the Glu 4 amide proton. The same amide proton signal was greatly intensified when hDOR-(281-297) was added to the sample (Fig. 8b). This result demonstrates a deshielding of the deltorphin II Glu 4 amide proton when encountering the hDOR peptide. A likely explanation for this event is the breaking of an internal hydrogen bond in deltorphin II involving Glu 4 NH. Indeed, some groups have reported deltorphin II adopting a U conformation that results in the N and C termini of the peptide approaching one another (32,33). In particular, a reverse turn at Val 5 has been proposed through NMR and modeling experiments (17). A hydrogen bond involving Glu 4 NH would stabilize this type of turn (34). Binding to hDOR could thus open up the deltorphin II U-shaped structure and lead to breaking of the hydrogen bond.
Upon shifting focus to the C-terminal end of the hDOR ECLIII, one notices a number of experimental pieces of evidence that explain events occurring in this region of the receptor following ligand binding. First of all, a ␤-structure at the C terminus of the hDOR ECLIII peptides was observed when the peptide sequence ended in Val 297 , but was absent when Ala 298 and Ala 299 were included. The NOEs observed between Asp 293 and Leu 295 and between Pro 294 and Val 296 for hDOR-(281-297) and hDOR-(283-297) suggest that formation of the ␤-structure is the result of a reverse turn centered on Pro 294 (35). A Chou-Fasman protein conformational profile plot was made for different variants of the C-terminal sequence after and including Arg 292 using the program Peptide Companion (CoshiSoft/Pep-tiSearch Version 1.25). A high propensity to adopt either a helical conformation or a ␤-conformation was noted after Pro 294 when Ala 298 and Ala 299 were present, whereas only a ␤-structure was calculated for this domain when the two alanines were removed (data not shown). Lengthening the C terminus with two additional alanines thus potentially allows for competing C-terminal secondary conformations that prevent a stable turn at Pro 294 . This would explain the results observed for the receptor peptides presented here.
It has been demonstrated that a hydrophobic segment including residues Leu 295 to Leu 300 in the intact receptor is an important element for ␦-selective ligand binding (21). Much supporting information for this was obtained from the results presented here. According to ␣-proton chemical shift measurements, hDOR-(279 -299) gained a C-terminal ␤-conformation (Fig. 7a) upon complexation with deltorphin II. In particular, the ␣-proton chemical shifts for the important residues Val 296 and Val 297 moved significantly upfield as a result of deltorphin II binding (Figs. 6 and 7a). A slow amide proton exchange with the surrounding water was also observed for hDOR-(281-297) residues Leu 295 , Val 296 , and Val 297 when deltorphin II was present, whereas only Val 296 NH appeared shielded from the aqueous environment in the absence of deltorphin II (Fig. 9). Likewise, the amide proton of Gly 7 in deltorphin II was protected from the solvent in the presence of hDOR-(281-297) (Fig. 9). One can conclude from these data that there is some hydrophobic interaction between Leu 295 , Val 296 , and Val 297 of hDOR-(281-297) and the C-terminal address domain of deltorphin II (deltorphin II Val 5 -Gly 7 ). Indeed, many groups have reported that the hydrophobicity associated with Val 5 and Val 6 is critical for ␦-opioid receptor binding and selectivity (13)(14)(15). Therefore, it is possible that Val 5 and Val 6 of deltorphin II interact with the Leu 295 -Val 297 segment of hDOR. Support for this hypothesis stems from the observed dramatic displacement of ␣-proton chemical shifts for Val 296 and Val 297 when deltorphin II was added to a sample of hDOR-(279 -299) (Figs. 6 and 7a).
When a Chou-Fasman calculation was repeated for the Arg 292 -Ala 299 segment of hDOR with alanine residues replacing the two valines, a helix appeared as the only possible conformation available to the Leu 295 -Ala 296 -Ala 297 -Ala 298 -Ala 299 sequence (data not shown). Mutating Val 296 or Val 297 to alanine experimentally also abolished deltorphin II binding, thus suggesting that a helical conformation in the important hDOR hydrophobic domain (Leu 295 -Leu 300 ) is unfavorable for deltorphin II binding. All of the evidence taken together indicates that a turn centered on Pro 294 may form following deltorphin II binding to hDOR Leu 295 -Leu 300 in an extended conformation.
The loss of a helical conformation unexpectedly also occurred for hDOR-(279 -299) following addition of the -selective peptides endomorphin-1 and -2. The residue sequences for these two peptides contain a Tyr-Xaa-Phe (or Tyr-Xaa-Trp in the case of endomorphin-1) motif commonly associated with the message component of opioid ligands (28). It is therefore possible that the endomorphins bind weakly to the ␦-opioid receptor at levels undetectable in conventional binding assays in the vicinity of Trp 284 , yet these peptides possibly lack the additional molecular determinants that form the address domain of ␦-selective ligands. When the -selective peptide agonist dynorphin A-(1-13) was allowed to interact with the hDOR ECLIII peptide, a very small stabilization of the helix was noted. Dynorphin A-(1-13) does not contain an N-terminal Tyr-Xaa-Phe motif like the other peptides studied, but rather a Tyr-Gly-Gly-Phe sequence. The CD data suggest that dynorphin A-(1-13) interacts very weakly with the hDOR ECLIII, but in a manner distinct from that of deltorphin II due to its additional Gly spacer in the message domain.
The small molecule -selective agonist U-50488 did not elicit a conformational change in the hDOR peptide. This was expected considering the small number of functional groups in the molecule and its established lack of binding to hDOR.
In conclusion, one may hypothesize conformational adjustments that accompany deltorphin II binding to the hDOR ECLIII. The spectroscopic evidence presented here suggests that Phe 3 and Glu 4 of deltorphin II interact with Trp 284 and Arg 291 of the ␦-opioid receptor, respectively, leading to destabilization of the helical structure at the N-terminal end of the receptor ECLIII. Coincidently, the important residues Val 5 and Val 6 in the address domain of deltorphin II interact with Val 296 and Val 297 at the C-terminal end of the hDOR ECLIII in an extended conformation and possibly induce a turn centered on Pro 294 .