Interaction of Extracellular Loop II of κ-Opioid Receptor (196–228) with Opioid Peptide Dynorphin in Membrane Environments as Revealed by Solid State Nuclear Magnetic Resonance, Quartz Crystal Microbalance and Molecular Dynamics Simulation

Abstract

κ-Opioid receptor is a member of the opioid receptor family and selectively interacts with the opioid peptide dynorphin. Extracellular loop II (ECL-II) of the κ-opioid receptor displays an amphiphilic helix in membrane environments and the N-terminal α-helix of dynorphin A(1-17) (hereafter DynA17) is inserted into the membrane with the tilt angle of 21° to the bilayer normal. ECL-II peptides (1-33), corresponding to 196-228 of κ-opioid receptor with [1-(13)C]- or [3-(13)C]-labeled amino acids were incorporated into large [dimyristoylphosphatidyl choline (DMPC)/ dihexanoylphosphatidyl choline (DHPC) = 3, q = 3] and small bicelle (q = 1) systems. (13)C direct detection with dipolar decoupling and magic angle spinning (DD-MAS) nuclear magnetic resonance (NMR) spectra were recorded, and the (13)C chemical shift perturbation clearly indicated that DynA17 interacts with ECL-II at the location of Val10-Ala15. Quartz crystal microbalance measurements were performed to determine the binding constant of ECL-II with DynA17 and indicated that the binding constant between DynA17 and ECL-II embedded in the lipid layer was 72 times larger than that between DynA17 and the lipid. The result of the molecular dynamics simulation clearly indicates that the C-terminus of DynA17 interact with the amino acid residues of the region between Val10-Gln14 of ECL-II. These results suggest that DynA17 interacts with the ECL-II of the κ-opioid receptor through a hydrophobic and short-lived electrostatic interaction with high affinity in the outer surface of the membrane.

Recent Solid-State NMR Studies of Membrane-Bound Peptides and Proteins

“…The results of MD simulations clearly indicated that the C-terminus of DynA17 interacts with the ECL-II residues in the region between Val10 and Gln14. These results suggest that DynA17 interacts with the ECL-II of the κ-opioid receptor through a hydrophobic and short-lived high-affinity electrostatic interaction in the outer surface of the membrane [77].…”

“…Solid-state NMR is particularly well suited for elucidating the dynamics, topology, orientation, and high-resolution structures of peptides in the bilayer environment using model and cell membranes. Peptides analyzed using this approach include venoms (melittin [27,29], bombolitin [54]), antimicrobial peptides (lactoferrampin [55], lactoferricin [56], PGLa [57][58][59], MSI-78 [60][61][62], LL-37 [63], pardaxin [62,64], magainin [65], P5 peptide [66], PG-1 [67], piscidin [68], crown ether containing 14-mer peptide [69,70]), K + channels (Shaker B ball peptide [71]), Neu receptor peptide [72], antibiotic peptides (alamethicin [73][74][75]), opioid peptides (dynorphin [76]), and κ-opioid receptor fragment (ECL-II [77]). In this section, the structure details of some of these peptides are reviewed in detail.…”

“…ECL-II peptides corresponding to residues 196-228 of the κ-opioid receptor and containing [1-13 C] or [3-13 C]-labeled amino acids were incorporated into large (DMPC/DHPC ¼ 3, q ¼ 3) and small (q ¼ 1) bicelle systems. 13 C direct detection with dipolar decoupling and magic-angle spinning (DD-MAS) NMR spectra was recorded, and the 13 C chemical-shift perturbation clearly indicated that DynA17 interacts with ECL-II at Val10-Ala15 [77]. Quartz-crystal microbalance measurements indicated that the binding constant between DynA17 and ECL-II embedded in the lipid layer was 72 times larger than that between DynA17 and the lipid [77].…”

“…13 C direct detection with dipolar decoupling and magic-angle spinning (DD-MAS) NMR spectra was recorded, and the 13 C chemical-shift perturbation clearly indicated that DynA17 interacts with ECL-II at Val10-Ala15 [77]. Quartz-crystal microbalance measurements indicated that the binding constant between DynA17 and ECL-II embedded in the lipid layer was 72 times larger than that between DynA17 and the lipid [77]. The results of MD simulations clearly indicated that the C-terminus of DynA17 interacts with the ECL-II residues in the region between Val10 and Gln14.…”

“…Another example of a membrane-embedded peptide is DynA17 which selectively binds to ECL-II of the κ-opioid receptor through Columbic interactions [77]. The orientation and dynamic structure of DynA17 in the membrane bilayer were determined using solid-state NMR spectroscopy [76,102].…”