Structure of the δ-opioid receptor bound to naltrindole

Abstract

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.

Figure 1. Overall structure of the δ-opioid receptor

(a) The δ-OR, orange, exhibits a typical seven-pass transmembrane architecture common to other GPCRs. (b-c) This fold is highly conserved among all three classical members of the opioid receptor family. (d) The opioid family possesses a conserved beta strand fold in the second extracellular loop (ECL2), creating a wide, open binding pocket. Despite the structural similarity, only five residues in this region are absolutely conserved. Conserved residues are highlighted red in sequence alignment and shown as sticks (e) ECL3, an important selectivity determinant for ligand binding, shows modest structural variability in the μ-OR and δ-OR. In the κ-OR receptor structure this region could not be resolved due to poor electron density. A single leucine residue is conserved among in ECL3 among opioid subtypes.

Figure 2. Ligand binding site of the δ-OR

Naltrindole binds in a deep but open binding site within the δ-OR. Selected contacts are highlighted at in (a), and a ligand F_o-F_c omit map within 2 Å radius of naltrindole is shown in (b) at a 3σ contour. The complete binding site is shown in (c) and (d). 2F_o-F_c electron density maps within a 2 Å radius of binding site amino acid side chains are shown in orange at a 1.5σ contour. The ligand omit density is shown as in (b).

Figure 3. A conserved opioid ligand recognition mode

(a) Opioid receptors bind a wide variety of ligands, including morphinans like naltrindole, other small molecules such as JDTic, and peptides like enkephalins. Most opioid ligands, including these, contain a “tyrosine” pharmacophore (red) with a phenolic hydroxyl in close proximity to a positive charge. Conserved recognition features for morphinan ligands observed in the μ-OR and δ-OR are highlighted on naltrindole (top). (b) The tyrosine pharmacophores of naltrindole, β-funaltrexamine, and JDTic are shown in their three dimensional context as observed in the crystal structures of δ-OR, μ-OR, and κ-OR. Specific conserved interactions in all three receptors are highlighted.

Figure 4. The message-address hypothesis is reflected in opioid receptor structure

Comparison of the δ-OR with κ-OR (a) and with μ-OR (b) reveals high conservation in both sequence and structure in the base of the ligand binding pocket, while extracellular regions are more divergent. Residue numbers and labels are those for δ-OR, with κ-OR residues in parentheses in (a) and μ-OR residues in parentheses in (b) where sequence differs from δ-OR. These regions interact with ligand moieties that can target binding to a particular opioid subtype. (c) Residues previously characterized as important for opioid subtype selectivity^- are clustered around the upper part of the binding pocket, delineating an “address” region of the receptor. (d) This region is structurally analogous to the allosteric site in muscarinic receptors (blue) suggesting that the high selectivity of muscarinic ligands occupying this space is also a manifestation of the message-address features structurally encoded within GPCRs.