(a) hKOR-T4L crystal packing. The parallel-dimer in one ASU is highlighted by insert. (b) Overall architecture of hKOR-T4L in complex with JDTic. A molecule (yellow) and B molecule (blue) in one ASU are aligned through the receptor part. The DRY and NPxxY motifs are highlighted in red and blue, respectively. JDTic is shown in a green sphere representation and the disulfide bonds are colored orange. (c) Side and (d) extracellular views of a structural alignment of hKOR (yellow); CXCR4 (PDB ID: 3ODU; magenta) and β₂AR (PDB ID: 2RH1; cyan). The graphics were created by PyMOL.

(a) Conformation of the binding pocket with JDTic shown by sticks with yellow carbons. The protein is displayed in cartoon representation looking down from the extracellular side, with the 22 contact residues within 4.5 Å from the ligand shown by white sticks. The pocket surface is shown as a semitransparent surface colored according to binding properties (green: hydrophobic, blue: H-bond donor, red: H-bond acceptor). Salt bridges and hydrogen bonds are shown as dotted lines. Structured water molecules are shown as large magenta spheres. (b) Diagram of ligand interactions in the binding pocket side chains at 4.5 Å cutoff. Salt bridges are shown in red and direct hydrogen bonds in blue dashed lines. Ballesteros-Weinstein numbering is shown as superscript. Residues that vary among MOR, DOR and KOR subtypes are highlighted in cyan, and residue Asp138^3.32 inferred in hKOR ligand binding by mutagenesis data, is highlighted orange. Side views of the sliced binding pocket in (c) hKOR-JDTic, (d) CXCR4-IT1t, and (e) β₂AR-carazolol complexes. The pocket surfaces are colored as in panel A, the protein interior is black and the extracellular space is white. Ligands are shown as capped sticks with carbons colored yellow (JDTic), magenta (IT1t) and cyan (carazolol). Asp^3.32 side chains in hKOR-JDTic and β₂AR-carazolol complexes are shown by thin sticks with grey carbons. The graphics were prepared using ICM molecular modeling package (Molsoft LLC).

Ligands are depicted as capped sticks with green carbons, and contact side chains of the receptor within 4 Å from the ligand are shown with grey carbons. Key hydrogen bonds and salt bridges are indicated with small cyan spheres and residues unique to KOR are labeled in blue. Residue Asp138^3.32, which also shows critical impact on GNTI and nor-BNI binding in mutagenesis studies, is highlighted red. Ballesteros-Weinstein residue numbers are shown under the hKOR residue numbers. The graphics were prepared using ICM molecular modeling package (Molsoft LLC).

Putative binding mode of (a) the RB-64 +463 amu and (b) the RB-64 +431 amu adduct. Residues within 4 Å of the ligand are displayed. Rendering: ligand, capped sticks/cyan carbons; hKOR side chains, capped sticks; hydrogen bonds, small green spheres; hKOR-unique residues labeled in blue. Ballesteros-Weinstein residue numbers are shown under the hKOR residue numbers. The graphics were prepared using ICM molecular modeling package (Molsoft LLC).