Receptor engineering to enable crystallization | Mouse receptor, with N- and C-terminal truncations; inserted N-terminal FLAG tag and C-terminal poly-His to aid purification; lysozyme T4L residues 2–161 inserted in ICL3; crystallized using lipidic cubic-phase technique with cholesterol | Mouse receptor, with N- and C-terminal truncations; inserted N-terminal FLAG tag and C-terminal poly-His to aid purification; lysozyme T4L residues 2–161 inserted in ICL3; crystallized using lipidic cubic-phase technique with cholesterol | Human receptor, with N- and C-terminal truncations; inserted N-terminal FLAG tag and C-terminal poly-His to aid purification; lysozyme T4L residues 2–161 inserted in ICL3; single point mutation I135L; crystallized using lipidic cubic-phase technique with cholesterol | Human receptor; replaced N terminus with a stabilized apocytochrome b-RIL fragment and a FLAG sequence; truncation of C terminus; crystallized using lipidic cubic-phase technique with cholesterol |
Cocrystallized ligand | β-FNA: MOPr-selective irreversible antagonist | Naltrindole: DOPr-selective reversible antagonist | JDTic: KOPr-selective reversible antagonist (Ki, 0.32 nM) | C-24: NOPr-selective reversible antagonist (Ki, 0.27 nM) |
TM domains and ECLs/ICLs (sequence homology data from Granier et al., 2012; Thompson et al., 2012) | 7TMs with similar placement to rhodopsin, with Pro-related bends in α-helices | 7TMs; 76% homology to MOPr; similar placement to rhodopsin, with Pro-related bends in α-helices | 7TMs; 73% homology to MOPr; ECL2 forms a β-hairpin | 7TMs; 67% homology to MOPr; similar placement to rhodopsin, with Pro-related bends in α-helices; ECL2 forms a β-hairpin; ECLs enriched in D, E residues; acidic relative to other OPrs; ICL2 forms a short α-helix |
Disulfide bridge | C140–C217; links ECL2 to end of TM3 | Not reported | C131–C210; links ECL2 to end of TM3 | C123(3.25)–C200(ECL2) |
Opioid ligand-binding pocket | “Open” binding pocket deep in cell membrane; should facilitate rapid dissociation of reversible ligands | “Open” binding pocket deep in cell membrane; similar to binding pocket in MOPr and KOPr | “Open” binding pocket deep in cell membrane; similar to binding pocket in MOPr and DOPr | Binding pocket is “relatively large” and capable of binding large peptides |
Critical ligand-binding residues | D147(3.32): charge-charge interaction with ligand; H297(6.52)+2H2O: hydrogen bonding to phenolic OH and aromatic ring of morphinans [ligand specific: K233(5.39): covalent link to β-FNA] | D128(3.32): charge-charge interaction with ligand; H278(6.52) +2H2O: hydrogen bonding to phenolic OH of naltrindole [probable ligand-specific roles for W274(6.48), Y308(7.43), M132(3.35), I277(6.51), Y129(3.33), V281(6.55), L300(7.35), W284(6.58)] | D138(3.32): charge-charge interaction with ligand; W287(6.48), H291(6.52): hydrophobic interactions with ligand [probable ligand-specific roles for V118(2.63), V134(3.28), L135(3.29), Y139(3.33), M142(3.36), V230(5.42), K227(5.39), I294(6.55), I290(6.51), Y312(7.35), I316(7.39), G319(7.42), V108(2.53), Q115(2.60), T111(2.56)] | D130(3.32): charge-charge interaction with ligand; other binding pocket residues show reduced homology with KOPr or MOPr, reflecting low affinity for classic opioids; H(6.52) replaced by Q280(6.52); M134(3.36) reoriented relative to M142(3.36) in KOPr; A216(5.39) replaces K; T305(7.39) replaces I in other OPrs |
Oligomerization | Crystallizes as parallel dimers; tightly associated through TM5, TM6 | Crystallizes as antiparallel dimers, possibly reflecting energetically favorable interactions unique to crystallization conditions | Crystallizes as parallel dimers; structures of the two molecules in the dimer are similar but not identical, for example in ICL2 | Not reported |
Reference | Manglik et al., 2012 | Granier et al., 2012 | Wu et al., 2012 | Thompson et al., 2012 |