ABSTRACT
The µ-opioid receptor (µOR) is an important target for pain management1 and molecular understanding of drug action on µOR will facilitate the development of better therapeutics. Here we show, using double electron-electron resonance and single-molecule fluorescence resonance energy transfer, how ligand-specific conformational changes of µOR translate into a broad range of intrinsic efficacies at the transducer level. We identify several conformations of the cytoplasmic face of the receptor that interconvert on different timescales, including a pre-activated conformation that is capable of G-protein binding, and a fully activated conformation that markedly reduces GDP affinity within the ternary complex. Interaction of ß-arrestin-1 with the µOR core binding site appears less specific and occurs with much lower affinity than binding of Gi.
Subject(s)
Ligands , Protein Conformation , Receptors, Opioid, mu , Humans , beta-Arrestin 1/chemistry , beta-Arrestin 1/metabolism , Binding Sites , Fluorescence Resonance Energy Transfer , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , Guanosine Diphosphate/metabolism , Guanosine Diphosphate/chemistry , Models, Molecular , Protein Binding , Receptors, Opioid, mu/metabolism , Receptors, Opioid, mu/chemistry , Single Molecule ImagingABSTRACT
The µ-opioid receptor (µOR) is an important target for pain management and the molecular understanding of drug action will facilitate the development of better therapeutics. Here we show, using double electron-electron resonance (DEER) and single-molecule fluorescence resonance energy transfer (smFRET), how ligand-specific conformational changes of the µOR translate into a broad range of intrinsic efficacies at the transducer level. We identify several cytoplasmic receptor conformations interconverting on different timescales, including a pre-activated receptor conformation which is capable of G protein binding, and a fully activated conformation which dramatically lowers GDP affinity within the ternary complex. Interaction of ß-arrestin-1 with the µOR core binding site appears less specific and occurs with much lower affinity than binding of G protein Gi.
ABSTRACT
The indanedione series of vinyl ketone spin-labelling reagents has been extended in two ways: by increasing the length of the rigid spacer between the reactive centre and the nitroxide ring, or by introducing an electrophilic substituent (that could also hinder its rotation) at the bridge head position of the nitroxide ring. Three reagents of this new series have been used to spin label the Class II thiol groups of membranous Na,K-ATPase from Squalus acanthias. With a conjugated diene spacer, the majority of spin labels are strongly held but a minor population is relatively mobile at 37 degrees C. With a conjugated triene spacer, the nitroxide is still strongly held but a portion of the label is non-covalently bound. The 4-bromo-pyrroline derivative (with short vinyl spacer) is tightly held at the attachment site, and the conventional electron paramagnetic resonance (EPR) spectra distinguish between the two enantiomeric structures which differ in their mobility at 37 degrees C. Saturation transfer EPR (ST-EPR) spectra of this label at 4 degrees C have been used to determine the dependence of the protein rotational mobility on ionic strength. Electrostatic repulsion contributes to the lateral interactions between Na,K-ATPase molecules.
