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, 288 (48), 34470-83

Chemotype-selective Modes of Action of κ-Opioid Receptor Agonists

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Chemotype-selective Modes of Action of κ-Opioid Receptor Agonists

Eyal Vardy et al. J Biol Chem.

Abstract

The crystal structures of opioid receptors provide a novel platform for inquiry into opioid receptor function. The molecular determinants for activation of the κ-opioid receptor (KOR) were studied using a combination of agonist docking, functional assays, and site-directed mutagenesis. Eighteen positions in the putative agonist binding site of KOR were selected and evaluated for their effects on receptor binding and activation by ligands representing four distinct chemotypes: the peptide dynorphin A(1-17), the arylacetamide U-69593, and the non-charged ligands salvinorin A and the octahydroisoquinolinone carboxamide 1xx. Minimally biased docking of the tested ligands into the antagonist-bound KOR structure generated distinct binding modes, which were then evaluated biochemically and pharmacologically. Our analysis identified two types of mutations: those that affect receptor function primarily via ligand binding and those that primarily affect function. The shared and differential mechanisms of agonist binding and activation in KOR are further discussed. Usually, mutations affecting function more than binding were located at the periphery of the binding site and did not interact strongly with the various ligands. Analysis of the crystal structure along with the present results provide fundamental insights into the activation mechanism of the KOR and suggest that "functional" residues, along with water molecules detected in the crystal structure, may be directly involved in transduction of the agonist binding event into structural changes at the conserved rotamer switches, thus leading to receptor activation.

Keywords: G Protein-coupled Receptors (GPCR); Mutagenesis Site-specific; Opiate Opioid; Pharmacology; Receptor Structure-Function.

Figures

FIGURE 1.
FIGURE 1.
KOR-selective agonists used in this study.
FIGURE 2.
FIGURE 2.
Effect of the different mutations on binding affinity and potencies of the tested ligands. Histogram bar heights represent the difference between the KOR mutant and WT KOR potency (ΔpIC50) and affinity (ΔpKi) of the tested ligands salvinorin A (red), 1xx (red with diagonal hash marks), U-69593 (light blue), and dynorphin A (dark blue). Asterisks indicate low expressing mutants; the effect on function is at least partially due to low expression levels.
FIGURE 3.
FIGURE 3.
Proposed binding mode for dynorphin A(1–8). A, putative interaction of Arg7 of dynorphin A with Glu297(6.58) probably disturbs the stability of a salt bridge formed between Lys227(5.39) and Glu297(6.58) (potential hydrogen bonds are indicated with green lines). This salt bridge may serve to attenuate G protein-mediated signaling by limiting the ability of TM6 to adopt an active-like conformation, an effect that is probably analogous to the formation of the Ser222(5.43)–Asn344(6.55) hydrogen bond (and other nonpolar interactions) upon binding of the β-arrestin-selective agonist ergotamine at the 5-HT2B receptor. B, putative binding mode showing KOR amino acid residues within 4 Å of the ligand. Water molecules W1–W4 correspond to Wat1311, -1307, -1316, and -1314, respectively.
FIGURE 4.
FIGURE 4.
Proposed binding mode for U-69593. A, putative interaction of U-69593 with Ile316(7.39) and Ile290(6.51), binding site residues at positions that belong to a conserved network of key GPCR non-covalent interactions. Mutation of either Ile316 or Tyr320(7.43) one turn above significantly reduces the potency of all tested agonists, consistent with the importance of Ile316 for proper GPCR structure and function. Structurally, a likely explanation for this effect is that these mutations significantly hinder the ability of TM7 and its NPXXY motif to adopt a G protein-recognizing conformation. A hydrogen bond (shown as a green line) may be formed between Tyr312(7.35) and the pyrrolidine ring nitrogen atom in an alternate ring puckering conformation. B, putative binding mode showing KOR amino acid residues within 4 Å of the ligand. Water molecules W1–W4 correspond to Wat1311, -1307, -1316, and -1314, respectively.
FIGURE 5.
FIGURE 5.
Proposed binding mode for 1xx. A, putative interaction of 1xx with Met142(3.36) at the bottom of the intrahelical ligand binding pocket. Mutation of Met142 to alanine adversely affects the potency of all tested agonists except 1xx, and the affinity of 1xx, unlike that of the other agonists, was substantially increased in the M142A mutant. Because Met142 is directly adjacent to and interacts extensively with the His291(6.52) and Trp287(6.48), its mutation to alanine disrupts the ability of the KOR to function. However, the putative binding mode of 1xx suggests that its bulky 4-chloro-3-trifluoromethyl group, located directly adjacent to Met142, could maintain the signaling ability of the M142A mutant by preventing collapse in the region caused by the missing methionine atoms. B, putative binding mode showing KOR amino acid residues within 4 Å of the ligand. Water molecules W1–W4 correspond to Wat1311, -1307, -1316, and -1314, respectively.
FIGURE 6.
FIGURE 6.
Proposed binding mode for salvinorin A. A, putative interaction of salvinorin A with, among other residues, Ile316(7.39) and Ile290(6.51) in the KOR orthosteric binding site. Like U-69593, the close association of the ligand with TM7 probably interferes with the ability of TM7 to adopt a G protein-recognizing conformation. B, putative binding mode showing KOR amino acid residues within 4 Å of the ligand. Water molecules W1–W4 correspond to Wat1311, -1307, -1316, and -1314, respectively.
FIGURE 7.
FIGURE 7.
The relationship between the effect of mutations on binding and function of the different ligands. The effect of mutations on binding affinity (ΔpKi) is compared with their effect on function (ΔpIC50) for the tested ligands salvinorin A, 1xx, dynorphin A, and U-69593. The middle diagonal line indicates identical effects on binding and function. The outside lines are 1 log unit above and below this “line of identical effect.” Gray points indicate mutations that affected function more than binding. Black points outside the 1 log difference line belong to the low expressing mutations L212A and I135A.
FIGURE 8.
FIGURE 8.
Proposed activation pathways in the κ-opioid receptor. A, an overall view of the JDTic-bound KOR crystal structure with the important rotamer switch motifs (green residues, DRY; orange residues, NPXXY; purple residues, P-I-F motif). B, focus on the interface between TM3, TM5, and TM6, showing the proposed structural relationship between some of the tested positions and the P-I-F motif. C, the predicted changes in the rotameric state of Ile(3.40) and Phe(6.44) (orange) upon activation (based on the analysis of the active state P-I-F motif in several GPCR structures) introduce a collision with the water molecule trapped between Ile146 and His291 in the KOR crystal structure, suggesting that this water molecule is displaced upon KOR activation. D, a surface representation of the binding site of KOR. Mutagenesis of Tyr139 can create a cleft in the wall of the binding site. Salvinorin A and 1xx are represented by thin white and yellow sticks, respectively.

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