Abstract
Allosteric modulators bear great potential to fine-tune neurotransmitter action. Promising targets are metabotropic glutamate (mGlu) receptors, which are associated with numerous brain diseases. Orthosteric and allosteric ligands act in synergy to control the activity of these multidomain dimeric GPCRs. Here, we analyzed the effect of such molecules on the concerted conformational changes of full-length mGlu2 at the single-molecule level. We first established FRET sensors through genetic code expansion combined with click chemistry to monitor conformational changes on live cells. We then used single-molecule FRET and show that orthosteric agonist binding leads to the stabilization of most of the glutamate binding domains in their closed state, while the reorientation of the dimer into the active state remains partial. Allosteric modulators, interacting with the transmembrane domain, are required to stabilize the fully reoriented active dimer. These results illustrate how concerted conformational changes within multidomain proteins control their activity, and how these are modulated by allosteric ligands.