Conformational dynamics of a class C G-protein-coupled receptor

smFRET is used to probe the activation mechanism of two full-length mammalian glutamate receptors, revealing that the extracellular ligand-binding domains of these G-protein-coupled receptors interconvert between three confirmations (resting, activated and a short-lived intermediate state), and that the efficacy of an orthosteric agonist correlates with the degree of occupancy of the active state. Metabotropic glutamate receptors (mGluRs) are dimeric class C G-protein-coupled receptors (GPCRs) that modulate neuronal excitability, synaptic plasticity, and serve as drug targets for neurological disorders such as schizophrenia and fragile X syndrome. There have been several X-ray crystal structures of GPCRs in the past few years, but our understanding of the conformational dynamics of receptor activation is incomplete. Here the authors used single-molecule fluorescence resonance energy transfer (smFRET) to probe the activation mechanism of two full-length mammalian mGluRs. The smFRET experiments revealed that the extracellular ligand-binding domains of these GPCRs interconvert between three conformations (resting/inactive, activated, and a short-lived, inactive intermediate state) and that efficacy of an orthosteric agonist correlates with the degree of occupancy of the active state. The experimental strategy described in this paper should be widely applicable to the study of conformational dynamics in GPCRs and other membrane proteins. G-protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors in eukaryotes. Crystal structures have provided insight into GPCR interactions with ligands and G proteins1,2, but our understanding of the conformational dynamics of activation is incomplete. Metabotropic glutamate receptors (mGluRs) are dimeric class C GPCRs that modulate neuronal excitability, synaptic plasticity, and serve as drug targets for neurological disorders3,4. A 'clamshell' ligand-binding domain (LBD), which contains the ligand-binding site, is coupled to the transmembrane domain via a cysteine-rich domain, and LBD closure seems to be the first step in activation5,6. Crystal structures of isolated mGluR LBD dimers led to the suggestion that activation also involves a reorientation of the dimer interface from a 'relaxed' to an 'active' state7,8, but the relationship between ligand binding, LBD closure and dimer interface rearrangement in activation remains unclear. Here we use single-molecule fluorescence resonance energy transfer to probe the activation mechanism of full-length mammalian group II mGluRs. We show that the LBDs interconvert between three conformations: resting, activated and a short-lived intermediate state. Orthosteric agonists induce transitions between these conformational states, with efficacy determined by occupancy of the active conformation. Unlike mGluR2, mGluR3 displays basal dynamics, which are Ca2+-dependent and lead to basal protein activation. Our results support a general mechanism for the activation of mGluRs in which agonist binding induces closure of the LBDs, followed by dimer interface reorientation. Our experimental strategy should be widely applicable to study conformational dynamics in GPCRs and other membrane proteins.