The NMR-Derived Conformation of Orexin-A: An Orphan G-Protein Coupled Receptor Agonist Involved in Appetite Regulation and Sleep

Abstract

The conformation of orexin-A, an orphan G-protein coupled receptor agonist has been determined when bound to sodium dodecylsulphate-d(25) (SDS) micelles by (1)H and (13)C NMR and molecular modeling. Orexin-A has been implicated in sleep-wakefulness and feeding regulation. The conformational preference of orexin-A consists of a short helical section, involving Asp(5) to Gln(9) that makes up helix I, followed by a bend from Lys(10) to Ser(13). Residues Leu(16) to Gly(22) make up helix II. The conformation of orexin-A can now be used to explain the results of earlier Ala substitution mutagenesis experiments (J. G. Darker et al., Bioorg. Med. Chem. Lett. 11, 737-740 (2001); S. Ammoun, et al., J. Pharmacol. Expt. Ther. 305, 507-514 (2003)). Darker et al., working with orexin-A (15-33) amide, observed a significant drop in functional potency at the OX(1)R receptor when Leu(16), Leu(19), Leu(20), His(26), Gly(29), Ile(30), Leu(31), Thr(32), and Leu(33) were replaced by Ala. Ammoun et al. identified three areas of interest, which were the same for OX(1)R and OX(2)R receptors, as amino acids 15-17, 20 and 25-26 with the most marked reduction in activity being produced by the replacement of Leu(20) by Ala. We suggest that Leu(16), Leu(19), and Leu(20), which are in helix II, are likely responsible for binding orexin-A to the surface of the micelle.

Recognition of GPCRs by Peptide Ligands and Membrane Compartments theory: Structural Studies of Endogenous Peptide Hormones in Membrane Environment

“…Due to spectral overlap and lack of many NOE restraints, it was difficult to determine the structure of functionally important C-terminal region in SDS micelles. Miskolzie and Kotovych [62] have predicted that this segment might adopt a turn-like or short helical structure. In contrast to the micelle structure, C-terminal region of orexin-A is clearly helical in solution (PDB codes: 1WSO and 1R02).…”

“…The absence of orexin peptides in the patient results in narcolepsy, a chronic sleep disorder [150]. Structures of both the orexin peptides have been determined in SDS micelles [52,62] as well as in solution [151].…”

Truncated Orexin Peptides: Structure–Activity Relationship Studies

“…NMR and computational studies performed on orexin A (in aqueous 25 and in membrane mimetic micellar solutions 26 ) identified a highly conserved hydrophobic region on the C-terminus. This region contains two α-helices that are connected by a short linker.…”

“…NMR and computational studies performed on orexin A (in aqueous and in membrane mimetic micellar solutions) identified a highly conserved hydrophobic region on the C-terminus. This region contains two α-helices that are connected by a short linker. , Similarly, solution structure of orexin B (in aqueous and in micellar solution) also shows two α-helices at similar positions.…”

“…In the proposed orexin A structure, the residues that were deemed important for potency in truncated peptide OXA (15–33) (Leu 16 , Leu 19 , Leu 20 , His 26 , Gly 29 , Ile 30 , Leu 31 , Thr 32 , and Leu 33 ) seemed to exist on one side of orexin A, forming a hydrophobic surface. , This surface might also be important in binding to the cell membrane . Residues that were less important (Arg 15 , Tyr 17 , Glu 18 , His 21 , Gly 22 , Gly 24 , and Asn 25 ) formed a hydrophilic surface on orexin A, which were thought to play a minimal role in potency.…”