Exploring deltorphin II binding to the third extracellular loop of the delta-opioid receptor.
The third extracellular loop of the human delta-opioid receptor (hDOR) is known to play an important role in the binding of delta-selective ligands. In particular, mutation of three amino acids (Trp(284), Val(296), and Val(297)) to alanine significantly diminished delta-opioid receptor affinity for delta-selective ligands. To assess the changes in conformation accompanying binding of the endogenous opioid peptide deltorphin II to the delta-opioid receptor at both the receptor and ligand levels as well as to determine points of contact between the two, an in-depth spectroscopic study that addressed these points was initiated. Fragments of the delta-opioid receptor of variable length and containing residues in the third extracellular loop were synthesized and studied by NMR and CD spectroscopy in a membrane-mimetic milieu. The receptor peptides examined included hDOR-(279-299), hDOR-(283-299), hDOR-(281-297), and hDOR-(283-297). A helical conformation was observed for the longest receptor fragment between Val(283) and Arg(291), whereas a nascent helix occurred in a similar region for hDOR-(281-297). Further removal of N-terminal residues Val(281) and Ile(282) abolished helical conformation completely. Binding of the delta-selective ligand deltorphin II to hDOR-(279-299) destabilized the helix at the receptor peptide N terminus. Dramatic changes in the alpha-proton chemical shifts for Trp(284) and Leu(286) in hDOR-(279-299) also accompanied this loss of helical conformation. Large upfield displacement of alpha-proton chemical shifts was observed for Leu(295), Val(296), and Val(297) in hDOR-(279-299) following its interaction with deltorphin II, thus identifying a gain in beta-conformation at the receptor peptide C terminus. Similar changes did not occur for the shorter peptide hDOR(281-297). A hypothesis describing the conformational events accompanying selective deltorphin II binding to the delta-opioid receptor is presented.
AstraZeneca Research and Development, 7171 Frédérick-Banting, Ville Saint-Laurent, Montréal, Québec H4S 1Z9, Canada.
Ibtihal
I
Fadhil
Ralf
R
Schmidt
Chris
C
Walpole
Katharine A
KA
Carpenter
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002942:Circular Dichroism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017465:Receptors, Opioid, delta / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D009842:Oligopeptides; D017465:Receptors, Opioid, delta; C060209:deltorphin II, Ala(2)-