Κ-Opioid Receptor General Structure

Κ-Opioid Receptor  Structure

The crystal structure of kOR-JDTic ligand complex at a resolution of 2.9 Å revealed this receptor is structurally very similar to receptors of the same γ subclass GPCRs and even other members of the broader class A GPCR family (See Figure 4).

All four opioid receptor subtypes share a GPCR-type structure with 7 alpha helices, which constitute the transmembrane domains, and a G_i/G₀ protein associated with an eighth intracellular α-helix that runs parallel to the membrane. They also contain three extracellular loops (ECLs) and three intracellular loops (ICLs). ECL2 is the largest of these receptor loops and contains a disulphide bond which is thought to stabilize a β-hairpin structure which covers a ligand binding pocket. Opioid receptors share approximately 70% sequence identity in the transmembrane regions and the C-terminus has a high degree of homology. Most of the sequence variation between opioid receptors is found in the ECLs, which are likely to play an important role in selective ligand binding. The crystal structure of the protein revealed k-OR to contain a disulphide bond between Cys131 and Cys 210 bridging ECL2 to the end of helix III. This last feature is present in all opioid receptors and is thought to be a common component of many class A GPCRs. Another canonical, class A GPCR, feature identified in the structure of the k-OR is a ‘NPXXY’ amino acid residue motif in helix VII where the ‘X’ residue varies between different class A GPCR proteins. In the case of k-OR this motif encompasses Asn326, Pro327, Ile328, Leu 329 and Tyr 330. This sequence is thought to be involved in class A GPCR activation by acting as a molecular switch. This particular region of the receptor was observed to have a relatively high structural similarity with β₂-adrenergic receptors and adenosine receptors (A₂AR).

 However, a number of structural characteristics were identified in the crystallographic study of k-ORs, which are believed to be unique and are likely to also be determinants of the selectivity of the ligand-receptor interactions.  One of these features is the disulphide bond interaction between ECL3 and the N-terminus of the protein which causes helix I to tilt towards the transmembrane bundle. Another example is how regions of the ECL3 seem to be disordered in some residue stretches, resulting in no interpretable electron density in these regions. The crystal structure in conjunction with mutagenesis studies also revealed an important inter-helical hydrogen bond between Arg156 and Thr273 (the latter in helix VI) which is important for the stabilization of the inactive conformation of k-ORs. The degree of conservation of the latter interaction throughout the class A GPCR family is still being assessed.

In a nutshell, the crystallographic study of k-OR revealed that the structure of this receptor was highly similar to that of members of the same GPCR subclass and even family, and so were the molecular interactions that held the structure together. This is not a surprise since GPCRs are a good example of how a range of functions evolved from a particular protein topology. The similarity of k-OR structure with that of proteins from the same family was high even though many of these regions contained a significant difference in amino acid sequence (mutagenesis experiments confirmed this where amino acid substitution for that present in other proteins such as the β₂-adrenergic receptor didn’t cause a significant change in structure or affinity). However the crystal structure did confirm that most of sequence diversity was present in the ECLs, where the ligand binding pocket was present. This suggested that the ability of the opioid ligands to distinguish between very similar receptors and selectively bind to them was likely to lie in the chemical properties of the receptor binding pocket and the chemical structure of the ligand itself, rather than the general structure of the k-OR, which is more of a stabilizing framework for this interaction to occur and for further signal transduction to be possible through interaction with intracellular G-proteins.

Figure 4. Structure of the opioid receptor classes. A) Structure of two dimerized kappa-opioid receptors with associated G-proteins. This is the assymetric subunit used to elucidate the structure of the receptor through X-ray crystallography.  B) Structure of the Mu opioid receptor bound to a morphinian antagonist (red). C) Structure of a delta opioid receptor bound to naltrindole (red). D)  Structure of Nociceptin opioid receptor in association with a peptide mimetic. Note that although the receptors do have a similar overall structure, especially in the transmembrane bundle, the ECL regions on the ligand binding sites can be seen to differ significantly.