The Interaction of Highly Helical Structural Mutants with the NOP Receptor Discloses the Role of the Address Domain of Nociceptin/Orphanin FQ

• Published in: Chemistry : a European journal 2005-03, Vol.11 (7), p.2061-2070
• Main Authors: Tancredi, Teodorico, Carrà, Giacomo, Guerrini, Remo, Arduin, Marika, Calò, Girolamo, Regoli, Domenico, Salvadori, Severo, Temussi, Piero Andrea
• Format: Article
• Language: English
• Subjects: conformation analysis; Models, Molecular; Mutation; NMR spectroscopy; Nociceptin; Nociceptin Receptor; nociception; Opioid Peptides - chemistry; Opioid Peptides - genetics; Opioid Peptides - physiology; peptides; Protein Structure, Secondary; receptors; Receptors, Opioid - chemistry; structure- activity relationships; Structure-Activity Relationship
• ISSN: 0947-6539; 1521-3765
• DOI: 10.1002/chem.200401095

Nociceptin is a heptadecapeptide whose sequence is similar to that of Dynorphin A, sharing a message domain characterized by two glycines and two aromatic residues, and a highly basic C‐terminal address domain but, in spite of these similarities, displays no opioid activity. Establishing the relative importance of the message and address domains of nociceptin has so far been hampered by its extreme conformational flexibility. Here we show that mutants of this peptide, designed to increase the helical content in the address domain, can be employed to explain the mode of interaction with the NOP receptor. Nociceptin analogues in which Ala residues are substituted with aminoisobutyric acid (Aib) show a substantial increment of activity in their interaction with the NOP receptor. The increment of biological activity was attributed to the well‐documented ability of Aib to induce helicity. Here we have verified this working hypothesis by a conformational investigation extended to new analogues in which the role of Aib is taken up by Leu. The NMR conformational analysis confirms that all Ala/Aib peptides as well as [Leu7,11]‐N/OFQ‐amide and [Leu11,15]‐N/OFQ‐amide mutants (N/OFQ=nociceptin/orphanin FQ) have comparable helix content in helix‐promoting media. We show that the helical address domain of nociceptin can place key basic residues at an optimal distance from complementary acidic groups of the EL2 loop of the receptor. Our structural data are used to rationalize pharmacological data which show that although [Leu11,15]‐N/OFQ‐amide has an activity comparable to those of Ala/Aib peptides, [Leu7,11]‐N/OFQ‐amide is less active than N/OFQ‐amide. We hypothesize that bulky residues cannot be hosted in or near the hinge region (Thr5‐Gly6‐Ala7) without severe steric clash with the receptor. This hypothesis is also consistent with previous data on this hinge region obtained by systematic substitution of Thr, Gly, and Ala with Pro. Helicity is crucial for nociceptin: Nociceptin is a peptide that modulates several biological functions, including pain threshold, morphine analgesia, food intake, anxiety, locomotor activity, and memory processes. We show that mutants of this peptide, designed to increase the helical content in the address domain, clarify the mode of interaction with the NOP receptor, as the conformation of the domain places key basic residues (blue side chains—see graphic) at optimal distances from complementary acidic groups (red side chains) of the recep