Structural Modelling and Mutation Analysis of a Nociceptin Receptor and its Ligand Complexes

Abstract

We recently modelled and proposed four ligand-bound conformations for a G-protein-coupled receptor, namely, forms I, II, III and IV, based on the 3D structure and functional evidences for rhodopsin. In this study, the same strategy was applied to a human nociceptin receptor (NR), in order to predict ligand-bound receptor structures. Additionally, site-directed mutagenesis studies were carried out to evaluate these structures. A Thr138Ala mutant demonstrated the same affinity for [Phe(1)Psi(CH(2)-NH)Gly(2)]nociceptin(1-13)NH(2) as the wild-type receptor; however, the affinity of this mutant for nociceptin was 20-fold lower than that of the wild type. A Ser223Ala mutation showed the same characteristics as those of the wild type. On the other hand, a Gln280Ala mutation reduced the affinity to nociceptin by more than 60-folds. These results suggested that a change in the conformation of NR following agonist binding did not accompany the rigid-body rotation of the sixth transmembrane segment that was reported for an adrenergic receptor and a kappa-opioid receptor. NR is potently activated not only by nociceptin but also a synthetic peptide, i.e. Ac-RYYRIK-NH(2), although the amino acid sequences of both these ligands are completely different. The model explains why both the ligands activate NR and shows that their receptor-bound conformations have similar 3D structures.

The nociceptin receptor (NOPR) and its interaction with clinically important agonist molecules: a membrane molecular dynamics simulation study

“…Previously there were reports on homology modeling of NOPR. 31,32 More recently there were reports on the homology modeling of active state NOPR and further molecular dynamics simulation studies for a short period. 33 However, no study has been previously conducted on the time dependent interactions between NOPR and agonists for long time using MDS.…”

“…These interactions indicate the importance of Asp130, which is in line with previous results. 31,32 Moreover, Phe4 (N-H) of the message domain was found to be interacting with Tyr131. In addition, Lys9 hydrogen bonded with Asp110 and Glu199.…”

“…Previously, it has been proposed that Asp130 is involved in a salt bridge interaction with the positively charged N-terminal region (FGGF -message domain) of the NOP peptide. 31,32 Since we have the knowledge that electrostatic interaction between crucial residues (Asp130-N-terminal of peptide) dominates the interaction, we analyzed the electrostatically favored coefficients. Four clusters have been reported in the electrostatically favored coefficients and each cluster contains a lot of members in it.…”

Nociceptin/Orphanin FQ Receptor Structure, Signaling, Ligands, Functions, and Interactions with Opioid Systems

“…As expected, the ligand-binding pocket is contained within the transmembrane helices, with residues from TM3, TM5, TM6, and TM7 interacting with the ligand in the binding pocket. Similarly, molecular modeling of the complex of the peptide agonist N/OFQ with homology models of the NOP receptor (Topham et al, 1998;Akuzawa et al, 2007;Daga and Zaveri, 2012) show that the N-terminal sequence F-G-G-F of N/OFQ binds deep in the transmembrane binding pocket, where the N-terminal amino group of N/OFQ makes an essential anchoring charge interaction with the conserved D130 3.32 (superscripts refer to the BallesterosWeinstein numbering of the TM helix residue), present in all the opioid receptors as well as in biogenic amine GPCRs (Fig. 2).…”

Cloning and characterization of the rhesus monkey nociceptin/orphanin FQ receptor

“…Mouledous et al (2000) Akuzawa et al, 2007). The rest of substituted amino acids were scattered in N-terminal, third extracellular segment and C-terminal segment.…”