NMR conformational studies of micelle-bound orexin-B: a neuropeptide involved in the sleep/awake cycle and feeding regulation.
The preferred conformation of orexin-B, an orphan G-protein coupled receptor agonist (the human sequence is RSGPPGLQGRLQRLLQASGNHAAGILTM-NH(2)) has been determined by (1)H and (13)C 2D NMR spectroscopy and molecular modeling. Orexin-B has been implicated in sleep-wakefulness and feeding regulation. The membrane mimetic, sodium dodecylsulphate-d(25) (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 Leu(7) to Ser(18) and helix II spans Ala(22) to Leu(26). 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 [Phe(1)]-orexin-B conformation in water solution by NMR and showed that helix II extends from Ala(23) to Met(28). The C-terminal dipeptide, Thr(27)-Met(28), is unstructured is SDS, whereas in water it forms the end of helix II. The lack of apparent structure for Thr(27)-Met(28) 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 Gly(24) to Met(28) or D-amino acid substitution from Ala(23) to Met(28) causes a significant reduction in the potency of orexin-B for both OX(1)R and OX(2)R receptors. We postulate that helix II is involved in membrane recognition, and its binding to the membrane is essential for Thr(27)-Met(28) to adopt the correct receptor-binding conformation.
Miskolzie
Mark
M
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada, T6G 2G2.
Lucyk
Scott
S
Kotovych
George
G
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009479:Neuropeptides / Q000737:chemistry*; D064446:Orexin Receptors; D000068797:Orexins; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012890:Sleep; D012967:Sodium Dodecyl Sulfate; D013113:Spin Labels
D002352:Carrier Proteins; C000596229:HCRT protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008823:Micelles; D009479:Neuropeptides; D064446:Orexin Receptors; D000068797:Orexins; D010455:Peptides; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D013113:Spin Labels; D012967:Sodium Dodecyl Sulfate; D000409:Alanine