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Journal of Biochemistry Advance Access originally published online on April 24, 2007
Journal of Biochemistry 2007 141(6):907-916; doi:10.1093/jb/mvm100
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© 2007 The Japanese Biochemical Society.

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

Natsuyo Akuzawa1, Shigeki Takeda1,* and Masaji Ishiguro2

1Department of Nano-Material Systems, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515; and 2Suntory Institute for Bioorganic Research, 1-1 Wakayamadai, Shimamoto, Osaka 618-8503, Japan

*To whom correspondence should be addressed. Tel/Fax: +81-277-30-1434, E-mail: stakeda{at}chem-bio.gunma-u.ac.jp

Received December 27, 2006; Accepted April 9, 2007


  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 [Phe1{Psi}(CH2-NH)Gly2]nociceptin(1-13)NH2 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-NH2, 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.

Key Words: G-protein-coupled receptor, nociceptin receptor, modelling, site-directed mutagenesis

Abbreviations: F/G-NC, [Phe1 {Psi}(CH2-NH)Gly2]nociceptin(1-13)NH2; G{alpha}i1, {alpha} subunit of inhibitory GTP-binding regulatory protein subtype 1; GPCR, G-protein-coupled receptor; G-protein, heterotrimeric GTP-binding regulatory protein; [35S]GTP{gamma}S, guanosine 5'-O-(Formula [35S]thiotriphosphate); NR, a human nociceptin receptor; NR-G{alpha}i, a fusion protein of a human nociceptin receptor and a bovine Gi1 protein {alpha} subunit; TM, transmembrane segment; PCR, polymerase chain reaction


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