Structure of the nociceptin/orphanin FQ receptor in complex with a peptide mimetic

Abstract

Members of the opioid receptor family of G-protein-coupled receptors (GPCRs) are found throughout the peripheral and central nervous system, where they have key roles in nociception and analgesia. Unlike the 'classical' opioid receptors, δ, κ and μ (δ-OR, κ-OR and μ-OR), which were delineated by pharmacological criteria in the 1970s and 1980s, the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP, also known as ORL-1) was discovered relatively recently by molecular cloning and characterization of an orphan GPCR. Although it shares high sequence similarity with classical opioid GPCR subtypes (∼60%), NOP has a markedly distinct pharmacology, featuring activation by the endogenous peptide N/OFQ, and unique selectivity for exogenous ligands. Here we report the crystal structure of human NOP, solved in complex with the peptide mimetic antagonist compound-24 (C-24) (ref. 4), revealing atomic details of ligand-receptor recognition and selectivity. Compound-24 mimics the first four amino-terminal residues of the NOP-selective peptide antagonist UFP-101, a close derivative of N/OFQ, and provides important clues to the binding of these peptides. The X-ray structure also shows substantial conformational differences in the pocket regions between NOP and the classical opioid receptors κ (ref. 5) and μ (ref. 6), and these are probably due to a small number of residues that vary between these receptors. The NOP-compound-24 structure explains the divergent selectivity profile of NOP and provides a new structural template for the design of NOP ligands.

Figure 1. Structural overview of the NOP receptor

(a) Structural overview of NOP colored gray and ECL2 colored red. The bound ligand C-24 is depicted as green sticks, and transparent disks highlight the EC and IC membrane boundaries (colored blue and red, respectively). (b) Structural superposition of NOP molecules ‘A’ and ‘B’, κ–OR¹ (PDB ID 4DJH), and CXCR4 (PDB ID 3ODU) colored gray, yellow, blue and orange, respectively. Compared with κ–OR, the EC portion of helix I from NOP is tilted inward towards the orthosteric pocket in a conformation that is similar to CXCR4. (c) Structural superposition of NOP molecules ‘A’ and ‘B’ and thermostabilized A_2AAR (PDB ID 3PWH) colored gray, yellow, and green, respectively, highlighting conformational differences between the ICLs.

Figure 2. The orthosteric ligand binding pocket

(a) Cartoon representation of NOP with its large orthosteric ligand binding pocket shown as a blue transparent surface. ECL2 is colored red in all subsequent figures. (b) Extracellular view of the pocket with bound C-24 depicted as green sticks. (c) Side view of C-24 in the binding pocket with yellow dashed lines highlighting hydrogen bonding interactions and salt bridges. (d) Sigma-A weighted 2|mFo|-|DFc| electron density map contoured at 1.0 σ (0.0173 e/ Å ³) around C-24 inside the ligand binding pocket. (e) Schematic representation of C-24 interactions with NOP (B) with labeled distances (Å). Residue labels are colored according to the effect on C-24 binding when replaced with alanine: Magenta labels on black background abolish C-24 binding; red labels result in ~10-fold decrease in affinity; green labeled residues slightly increase the affinity of C-24, blue labeled residues were not tested, and Asp110 had no effect on the binding of C-24, although it is crucial for N/OFQ binding.

Figure 3. Molecular docking in the orthosteric binding pocket

Docking of C-24 (a), its analog C-35 (b), and peptide antagonist UFP-101 (c, d, e) in the NOP. The crystallographic pose of C-24 is green in all panels, and the docked molecules (C-24, C-35, UFP-101) are colored yellow. The Nphe1-Gly2-Gly3-Phe4 tetrapeptide portion of the docked UFP-101 is depicted as sticks, and the “address” domain (residues 5–17) of this peptide is represented as a cartoon. Panel (c) shows a ‘sliced’ side-view of the pocket; panel (d) shows a view from the extracellular surface; and panel (e) shows the electrostatic surface potentials of the UFP-101 peptide colored blue to red, corresponding to positive and negative surface potentials (+3 to −3 kT/e), respectively. ECL2 is colored red, and the acidic Asp and Glu residues from the ECL2 β-hairpin are depicted as red sticks.

Figure 4. Conformational differences in the ligand binding pocket between NOP/C-24 and κ–OR/JDTic

(a) ‘Sliced’ surface representation of NOP highlighting the deep binding pocket bound with C-24 (colored green) and JDTic (colored magenta) from the superimposed κ–OR structure. (b) (c) Different views of NOP (colored gray with green C-24) superimposed with the κ–OR structure (PDB ID 4DJH; colored blue with magenta JDTic). Hydrogen bonding interactions are depicted as dashed yellow and black lines for NOP and κ–OR, respectively. The waters from the κ–OR structure are depicted as cyan spheres. Residue labels are colored black and blue for NOP and κ–OR, respectively. Panel (a) highlights the conformational shifts observed between helices V and VI that result in differential binding pocket architectures. Panel (b) highlights the alternate rotamer of Met^3.36 (134 in NOP & 142 in κ–OR) in the pocket which affects the orientation of the ligand’s head groups.