Angiotensin II vs its type I antagonists: conformational requirements for receptor binding assessed from NMR spectroscopic and receptor docking experiments.

The conformations of three angiotensin II (AII) peptide antagonists ([Sar1]-AII(1-7)-NH(2), [Sar1,Val5,Ala8]-AII and the AII antipeptide, [Glu1,Gly2,Val5,Val8]-AII) were assessed in a lipid medium. A common backbone turn was identified through modeling and spectroscopic studies. The His6 residue acted as a pivoting point beyond which each peptide adopted two distinct conformations. One principle conformer resembled that previously determined for AII while the other was designated as an AII antagonist like conformer. A computational overlay between the nonpeptide antagonist, Losartan, and both the AII and the AII like conformation of [Sar1,Val5,Ala8]-AII revealed common pharmacophoric points with RMS deviations between 1 and 1.5 A. Both the AII conformer and the AII antagonist like conformer of [Sar1,Val5,Ala8]-AII were docked into a model of the AT(1) receptor. Receptor residue Phe289 and Asp281 provided good contact points for both peptides. Some differences were also noted. The terminal carboxyl of AII contacted Lys199 of the receptor while that of [Sar1,Val5,Ala8]-AII bridged Arg23 at the top of helix 1. The Asp1 side chain of AII interacted with His183 of the receptor.

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.