The Nociceptin Pharmacophore Site for Opioid Receptor Binding Derived from the NMR Structure and Bioactivity Relationships

Irina Nesmelova,Balazs Hargittai,Mary Pat Beavers,Michael J Orsini,Jingchun Liu,Steven A Middleton,Kevin H Mayo,Helen C Young,Peter J Connolly

doi:10.1074/jbc.m406405200

Irina Nesmelova, Balazs Hargittai + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m406405200

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Abstract

Nociceptin, a 17 amino acid opioid-like peptide that has an inhibitory effect on synaptic transmission in the nervous system, is involved in learning, memory, attention, and emotion and is also implicated in the perception of pain and visual, auditory, and olfactory functions. In this study, we investigated the NMR solution structure of nociceptin in membrane-like environments (trifluoroethanol and SDS micelles) and found it to have a relatively stable helix conformation from residues 4-17 with functionally important N-terminal residues being folded aperidoically on top of the helix. In functional assays for receptor binding and calcium flux, alanine-scanning variants of nociceptin indicated that functionally important residues generally followed helix periodicity, consistent with the NMR structural model. Structure-activity relationships allowed identification of pharmacophore sites that were used in small molecule data base searches, affording hits with demonstrated nociceptin receptor binding affinities.

Highlights

Nociceptin, a 17 amino acid opioid-like peptide that has an inhibitory effect on synaptic transmission in the nervous system, is involved in learning, memory, attention, and emotion and is implicated in the perception of pain and visual, auditory, and olfactory functions
As part of defining the best solution conditions for solution structure studies of nociceptin, Pulsed field gradient (PFG) NMR self-diffusion measurements were performed in water (D2O), in a water/TFE mixture, and in the presence of SDS micelles over the concentration range of 0.1–14 mM
Structure-Function Relations in Nociceptin effect is attributable to self-association of the peptide, and the magnitude drop in the diffusion coefficient is consistent with nociceptin forming dimers

Summary

Introduction

Nociceptin, a 17 amino acid opioid-like peptide that has an inhibitory effect on synaptic transmission in the nervous system, is involved in learning, memory, attention, and emotion and is implicated in the perception of pain and visual, auditory, and olfactory functions. Dynorphin A [25] and nociceptin [26] show little tendency to form well defined structures in water and in other polar solvents Because these peptides function by interacting with a membrane receptor, possibly by first interacting with membrane lipids [27], we investigated the NMR solution conformation of nociceptin in a relatively low dielectric environment, i.e. 30% (v/v) trifluoroethanol (TFE) and water, and in the presence of SDS micelles. Both systems are often employed in NMR investigations to mimic membrane-like environments. Tel.: 612-625-9968; Fax: 612-624-5121; E-mail: mayox001@ maroon.tc.umn.edu

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