Structure of the human k-opioid receptor in complex with JDTic

The human 𝛋-opioid receptor (𝛋-OR), along with μ and δ, belongs to the ɣ-subtype of opioid receptors within class A GPCR’s.

𝛋-OR mediates the effects of several neuropsychiatric pathways by activating inhibitory G-proteins. It is hence found in areas of the brain associated with cognition, mood and reward states (Carroll and Carlezon, 2013). The three OR subtypes share 70% sequence identity, indicating their similarity and conservation of key residues. Like all GPCRs, 𝛋-OR consists of a seven-transmembrane (TM) alpha-helical bundle structure, with an eighth helix running parallel to the membrane’s cytoplasmic side.

The crystal structure used in our figures, (PDB 4DJH) was determined by x-ray crystallography after 𝛋-OR was fused with T4 lysozyme for stabilisation purposes. It crystallised in complex with JDTic as a parallel dimer with interactions between helices I, II and VIII.

JDTic antagonises 𝛋-OR by blocking U50,488, a natural agonist. We discuss how JDTic specifically interacts with the receptor and how it does not affect μ and δ-OR’s despite their structural similarities.

General structure and binding pocket

Distinct features of 𝛋-OR were determined by comparison to other GPCR’s, such as CXCR4, which belongs to the ɣ-subfamily but binds peptides. A disulphide bond at the extracellular end of CXCR4’s helix I, and which is absent in 𝛋-OR, pulls it relatively closer to the TM helix bundle. However, both have a β-hairpin in their second extracellular loops which partially block their binding pocket entrances, this is despite low primary structure similarities in these regions.

𝛋-OR’s binding pocket is larger and deeper due to an inward shift of helix VI, and a has different set of side chains lining it.

Figure 1: 𝛋-OR has just one disulphide bond formed between Cys 131 and Cys 210 – appending ECL2 to Helix III. These two cysteines are conserved in all opioid receptors. Residues 120 to 130 were removed for ease of viewing.

JDTic selectivity

The D/ERY (Asp/Glu, Arg, Tyr) motif, which is found at the end helix III, forms a structure akin to that of rhodopsin and other GPCRs’ ionic lock (Chien et al, 2011). The arginine residue of D/ERY forms a hydrogen bond to Thr273 and stabilises the inactive conformation. Asn326, Pro, 327, Ile328, Leu329 and Tyr330 constitute the NPXXY motif located at the cytoplasmic end of helix VII, which is a highly conserved molecular switch responsible for activation. This was found to have a similar to conformation to that of A_2AAR in its inactive state, indicating 𝛋-OR assumes an inactive state in complex with JDTic.

 Figure 2: A depiction of the hydrogen bond formed between Arg 155 of helix III and Thr 273 of helix VI constituting 𝛋-OR’s ionic lock-like structure.

The positioning of JDTic deep in the binding cleft, facilitates the formation of ionic, polar and hydrophobic interactions.

The two most critical features of JDTic for its specific binding are its protonated amines of its piperidine and isoquinoline groups, as they form salt bridges with a conserved Asp138 (See Figure 3). 

These interactions force JDTic to assume V-shape conformation.

Figure 3

Highlights how Asp138 interacts with both 
piperidine and isoquinoline of JDTic.

Interestingly, Asp138 also forms ionic interactions with the equivalent groups to JDTic's piperidine, found in the nor-BNI and GNTI antagonists. (Munro et al, 2012) The complexes formed by k-OR with any of these three drugs have been shown to activate a protein kinase that suppresses any receptor signalling. The pathway which this happens will not be thoroughly discussed, but it occurs through blocking U50,488-k-OR complex formation and hence prevents kinase phosphorylation. (Bruchas & Chavkin, 2010) Evidence from mutagenesis studies have shown Asp138 is responsible for interacting with an amine group (see figure 3) (Subramanian et al, 1998), and it is true that all ligands for all opioid receptors contain amine groups.(Strahs & Weinstein, no date) Hence, this aspartate residue is conserved in all opioid receptors. 

Removing the distal hydroxyl groups of piperidine and isoquinoline moieties of JDTic, prevents water mediated interactions between them and 𝛋-OR, causing a 100-fold reduction in affinity.

𝛋-OR’s subtype selectivity is characterised by four residues, the combination of which differ across 𝛋, δ and μ (see Table 1 and figure 4).

𝛋	μ	δ
Val 108	Ala	Ala
Val 118	Asp	Lys
Ile 294	Val	Val
Tyr 312	Trp	Leu

Table 1: Relative residue changes between subtypes at particular locations in the 𝛋-OR sequence.

Where Val 118 is replaced with the larger Asp or Lys residues, unfavourable interactions are likely to be formed with the ligand, whilst replacement of Tyr 312 with tryptophan or leucine leads to a loss of an important polar interaction with JDTic’s amide. Val108 and Ile294’s replacement with alanine and valine respectively, is conducive to a reduction in hydrophobic interactions. All of the aforementioned substitutions are essential in the selectivity between opioid receptors.

Figure 4 shows the positions of the amino acid residues that were shown to be important for affinity of JTDic.
To see this in 3D space, follow this link

Hydrophobic Interactions

The Isopropyl group of JDTic forms hydrophobic interactions with a conserved Trp287, (see figure 5) which is part of an aromatic cluster and thought to play a key role in the activation mechanism of class A GPCRs. The hydrophobic interaction is highly energetically favourable and therefore blocks any conformational changes from occurring that may lead to the active conformation being adopted by the receptor. (Wu et al, 2012)

Figure 5

The hydrophobic interaction occurs between

the highlighted Trp287 residue and the

isopropyl group of JDTic. ECL2 is highlighted to

emphasise how deep into the binding pocket this hydrophobic interaction is.