NMR Conformational Studies of Micelle-Bound Orexin-B: A Neuropeptide Involved in the Sleep/Awake Cycle and Feeding Regulation

Abstract

The preferred conformation of orexin-B, an orphan G-protein coupled receptor agonist (the human sequence is RSGPPGLQGRLQRLLQASGNHAAGILTM-NH2) has been determined by 1H and 13C 2D NMR spectroscopy and molecular modeling. Orexin-B has been implicated in sleep-wakefulness and feeding regulation. The membrane mimetic, sodium dodecylsulphate-d25 (SDS), was used to mimic a physiological environment for the peptide. The secondary structure of orexin-B in SDS consists of two helical sections; helix I spans Leu7 to Ser18 and helix II spans Ala22 to Leu26. Helices I and II are believed to be involved in membrane binding, as is supported by the results of the spin label studies with 5-doxylstearic acid. Lee et al. (Eur. J. Biochem. 266, 831–839 (1999)) determined the [Phe1]-orex- in-B conformation in water solution by NMR and showed that helix II extends from Ala23 to Met28. The C-terminal dipeptide, Thr27-Met28, is unstructured is SDS, whereas in water it forms the end of helix II. The lack of apparent structure for Thr27-Met28 in SDS allows the dipeptide to have conformational freedom to interact with the receptor. The conformation of orexin-B can now be used to explain the Ala substitution mutagenesis experiments and the D-amino acid substitution experiments (S. Asahi et al, Bioorg. Med. Chem. Lett. 13, 111–113, 2003). Asahi et al. have shown that Ala substitution from Gly24 to Met28 or D- amino acid substitution from Ala23 to Met28 causes a significant reduction in the potency of orexin-B for both OX1R and OX2R receptors. We postulate that helix II is involved in membrane recognition, and its binding to the membrane is essential for Thr27-Met28 to adopt the correct receptor-binding conformation.