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Structure of the δ-opioid receptor bound to naltrindole

The opioid receptor family comprises three members, the µ-, δ- and κ-opioid receptors, which respond to classical opioid alkaloids such as morphine and heroin as well as to endogenous peptide ligands like endorphins. They belong to the G-protein-coupled receptor (GPCR) superfamily, and are excellent therapeutic targets for pain control. The δ-opioid receptor (δ-OR) has a role in analgesia, as well as in other neurological functions that remain poorly understood. The structures of the µ-OR and κ-OR have recently been solved. Here we report the crystal structure of the mouse δ-OR, bound to the subtype-selective antagonist naltrindole. Together with the structures of the µ-OR and κ-OR, the δ-OR structure provides insights into conserved elements of opioid ligand recognition while also revealing structural features associated with ligand-subtype selectivity. The binding pocket of opioid receptors can be divided into two distinct regions. Whereas the lower part of this pocket is highly conserved among opioid receptors, the upper part contains divergent residues that confer subtype selectivity. This provides a structural explanation and validation for the 'message-address' model of opioid receptor pharmacology, in which distinct 'message' (efficacy) and 'address' (selectivity) determinants are contained within a single ligand. Comparison of the address region of the δ-OR with other GPCRs reveals that this structural organization may be a more general phenomenon, extending to other GPCR families as well.

differences in the disordered amino and carboxy termini. The final crystallization construct (δ-OR-T4L) is shown in Supplementary Fig. 1.
The δ-OR-T4L construct was expressed in Sf9 cells using the pFastBac (Invitrogen) baculovirus system in the presence of 10 μM naloxone. Cell cultures were grown to a density of 4×10 6 cells per ml, infected with baculovirus containing the δ-OR-T4L gene, shaken at 27°C for 48 hr, and cell pellets were harvested and stored at -80 °C. To purify the protein, insect cells were first lysed by osmotic shock in a buffer comprised of 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 µM naltrindole and 2 mg/ml iodoacetamide to block reactive cysteines. This was followed by an extraction step, in which Sf9 membranes were homogenized with a glass dounce homogenizer in a solubilization buffer comprised of 1.0% lauryl maltose neopentyl glycol (MNG), 0.3% sodium cholate, 0.03% cholesterol hemisuccinate (CHS), 20 mM HEPES pH 7.5, 0.5M NaCl, 30% v/v glycerol, 2 mg/ml iodoacetamide, and 1 µM naltrindole. This extraction reaction was mixed at 4 °C for 1 hr, then centrifuged at high speed to remove cell debris. Nickel-NTA agarose was then added to the supernatant and stirred for 2 hr. The Nickel-NTA  Fig. 6).

Crystallization and data collection
Purified δ-OR-T4L receptor was concentrated to 50 mg/ml using a Vivaspin sample concentrator with a 50 kDa molecular weight cut-off (GE Healthcare). As for other GPCR-T4L fusion proteins crystallized to date, we utilized the in meso method to obtain crystals of δ-OR-T4L. Briefly, δ-OR-T4L was reconstituted into a mixture of monoolein and cholesterol (Sigma) by the two-syringe method. By weight, one part δ-OR-T4L was mixed with 1.5 parts of a 10:1 mixture of monoolein:cholesterol until the resulting phase was optically transparent. We used a Gryphon LCP robot (Art Robbins Instruments) to accurately dispense 20 to 55 nl mesophase drops onto glass plates. These lipidic boluses were overlaid with 700 nl precipitant solution. Crystals grew in precipitant solution consisting of 29-33% PEG 400, 100 mM HEPES pH 7.5, 120-180 mM sodium citrate (tribasic) and 350 mM magnesium chloride. Crystals were observed after 2 hrs and grew to full size after 5 days. Crystals used for data collection are shown in Supplementary Fig. 7.
Diffraction data were collected at Advanced Photon Source GM/CA-CAT beamline 23ID-B using a beam size of 10 μm. Due to radiation damage, the diffraction quality decayed during exposure. Wedges of 5 -15 degrees were collected and merged from 20 crystals using HKL2000 21 . Diffraction quality ranged from 3.0 -3.5 Å in most cases. Due to anisotropic diffraction (see Supplementary Table 1) the highest shell <I>/<I> value was slightly lower than is typical for isotropically diffracting crystals.
The structure of the δ-OR was solved by molecular replacement in Phaser 22 using the µ-OR receptor as a search model. The lattice for δ-OR-T4L shows alternating lipidic and aqueous layers, with receptor molecules arranged in anti-parallel associations ( Supplementary Fig. 8). We improved the initial model by iteratively building regions of the receptor in Coot 23 and refining in Phenix 24 .
To assess the quality of the final structure, we used MolProbity 25 . As with μ-OR and κ-OR, electron density was clear, and allowed confident placement of the ligand and binding site residues (Supplementary Fig. 9). The resulting statistics for data collection and refinement are shown in Supplementary