G protein-coupled receptors (GPCRs) relay information across cell membranes through conformational coupling between the ligand-binding domain and cytoplasmic signaling domain. In dimeric class C GPCRs, the mechanism of this process, which involves propagation of local ligand-induced conformational changes over 12 nm through three distinct structural domains, is unknown. Here, we used single-molecule FRET and live-cell imaging and found that metabotropic glutamate receptor 2 (mGluR2) interconverts between four conformational states, two of which were previously unknown, and activation proceeds through the conformational selection mechanism. Furthermore, the conformation of the ligand-binding domains and downstream domains are weakly coupled. We show that the intermediate states act as conformational checkpoints for activation and control allosteric modulation of signaling. Our results demonstrate a mechanism for activation of mGluRs where ligand binding controls the proximity of signaling domains, analogous to some receptor kinases. This design principle may be generalizable to other biological allosteric sensors.
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The custom codes for data analysis are available from the corresponding author upon reasonable request.
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We thank M.R. Schamber, D. Badong, D. May and A.Y. Pen for technical assistance, M. Gallio, J. Marko, A. Mondragon and K. Ragunathan for critical reading of the manuscript, and J. Fei (University of Chicago) for providing MATLAB scripts. This work was supported by the National Institutes of Health grant R01GM140272 (to R.V.) and by The Searle Leadership Fund for the Life Sciences at Northwestern University and by the Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust (to R.V.). B.W.L. was supported by the National Institute of General Medical Sciences (NIGMS) Training Grant T32GM-008061. This work used resources of the Keck Biophysics Facility supported in part by the NCI CCSG P30 CA060553 grant awarded to the Robert H. Lurie Comprehensive Cancer Center of Northwestern University.
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a, Schematic showing site-specific fluorescent labeling of mGluR2, with the unnatural amino acid 4-azido-L-phenylalanine at residue 548, by copper-catalyzed azide-alkyne click reaction. b, Donor (green: Cy3) and acceptor (red: Cy5) fluorophores conjugated to the CRD of inactive mGluR5 (PDB 6N52) at the position corresponding to residue 548 in mGluR2 (top). Representative confocal microscope image of HEK293T cells expressing 548UAA with the cell surface population labeled with donor (green: Cy3) and acceptor (red: Cy5) fluorophores through click chemistry (bottom). Scale bars, 10 µm. c, Unprocessed image of non-reducing 4-20% polyacrylamide gel electrophoresis of cell lysate from HEK293T cells expressing 548UAA and labeled by Cy5-alkyne. The gel is imaged with 633 nm excitation wavelength and 670-BP30 emission filter. Lane A: protein ladder; lane B: cell lysate; lane C: Cy5-alkyne dye. Results are representative of an individual experiment. d, Image of HEK293T cells expressing 548UAA and labeled with donor (green: Cy3) and acceptor (red: Cy5) fluorophores through click chemistry during live-cell FRET experiments. Scale bars, 10 µm. Results are representative of all titration and max response experiments for the 548UAA construct (N=21 independent experiments).
Extended Data Fig. 2 Live-cell ensemble FRET response to orthosteric ligands and a negative allosteric modulator.
a, Representative donor and acceptor intensities and corresponding FRET signal from live-cell FRET titration experiments for DCG-IV, LY379268 and Glutamate +10 µM Ro64-5229 measured using HEK293T cells expressing 548UAA (top) and corresponding dose-response curves (bottom). Each titration curve is normalized to the 1 mM glutamate response. Data represent mean ± s.e.m. of N=20, 10 and 13 cells for LY379268, DCG-IV and glutamate +10 µM Ro64-5229, respectively, examined over 3 independent experiments. b, Donor and acceptor intensities and corresponding FRET signal in response to saturating concentrations of glutamate (1 mM), DCG-IV (100 µM) and LY379268 (20 µM) in HEK293T cells expressing 548UAA.
a, Schematic of the single-molecule experiments (top). Representative frame from a single-molecule movie with the donor channel (Cy3) on the left and acceptor channel (Cy5) on the right (bottom). Molecules selected by analysis software for downstream processing are indicated by green circles. Scale bar, 3 µm. b, Example single-molecule time traces of the 548UAA in the absence of glutamate (0 µM) showing donor (green) and acceptor (red) intensities and corresponding FRET (blue). Dashed lines represent 4 distinct FRET states. c, Schematic of the VFT domain conformational sensor (left). Example single-molecule time traces of VFT domain sensor in the absence of glutamate (0 µM) and presence of saturating glutamate (1 mM) showing donor (green) and acceptor (red) intensities and corresponding FRET (blue) (bottom). smFRET population histogram of VFT domain mGluR2 sensor in the inactive (0 µM glutamate; 36 total molecules) and fully active (1 mM glutamate; 24 total molecules) conditions (top right). Data represent mean of N=2 independent experiments.
Extended Data Fig. 4 Example single-molecule time traces of CRD at different glutamate concentrations.
a, Example single-molecule time traces of the 548UAA at intermediate glutamate concentrations. b, Example single-molecule time traces of the 548UAA in saturating glutamate (1 mM). Donor (green) and acceptor (red) intensities and corresponding FRET (blue) are shown. Dashed lines represent 4 distinct FRET states.
a, smFRET population histograms of mGluR3 CRD sensor (labeled at residue 557) in the presence of competitive antagonist (LY341495; 221 total molecules) or saturating glutamate (290 total molecules). Data represent mean ± s.e.m. of N=3 independent experiments. Histograms are fitted (red) to 4 Gaussian distributions (blue) centered around 0.25 (state 1), 0.38 (state 2), 0.7 (state 3) and 0.87 (state 4), denoted with dashed lines. b, Example single-molecule time traces of mGluR3 CRD sensor in the presence of antagonist (LY341495) or saturating glutamate (1 mM) showing donor (green) and acceptor (red) intensities and corresponding FRET (blue). Dashed lines represent 4 distinct FRET states. Data was acquired at 100 ms time resolution.
Representative live-cell FRET measurement using HEK293T cells expressing 548UAA with the crosslinking mutation L521C upon application of intermediate (4 µM) and saturating glutamate (1 mM). FRET signal is normalized using initial FRET at time = 0.
a, Example single-molecule time traces of 548UAA at intermediate concentrations of DCG-IV (100 nM, top) and LY379268 (2 nM, bottom) showing donor (green) and acceptor (red) intensities and corresponding FRET (blue). Dashed lines represent 4 distinct FRET states. b, smFRET population histograms for 548UAA in the presence of saturating glutamate (1 mM; 152 total molecules), DCG-IV (100 µM; 470 total molecules) and LY379268 (20 µM; 356 total molecules). Data represent mean ± s.e.m. of N=3 independent experiments.
Heatmap illustrating the FRET values sampled by individual 548UAA receptors in the inactive (0 µM glutamate, top) and fully active (1 mM glutamate, bottom) conditions. Each row is the smFRET time trace of a single molecule over 6 seconds. The smFRET traces were smoothed using a 3-point moving average filter. 100 independent molecules are shown for each condition.
Example single-molecule time traces of 548UAA YADA/WT heterodimers at 20 µM glutamate. ATTO488 (light blue), donor (green) and acceptor (red) intensities and the corresponding FRET (blue) are shown. Dashed lines represent 4 distinct FRET states. Data was acquired at 100 ms time resolution.
a, smFRET population histograms of 548UAA PAM mutant (C770A) in the presence of 5 µM (689 total molecules) or 10 µM (370 total molecules) glutamate. Histograms are fitted (red) to 4 Gaussian distributions (blue) centered around 0.31 (state 1), 0.51 (state 2), 0.71 (state 3) and 0.89 (state 4), denoted with dashed lines. Data represent mean ± s.e.m. of N=3 independent experiments. b, Normalized histograms of state 3 (pre-active) and state 4 (active) dwell times (cumulative count) in the presence of 5 or 10 µM glutamate for 548UAA and 548UAA PAM mutant (C770A). Data is fit to a single exponential decay function. Dwell times are from > 80 total molecules per condition from two independent experiments. c, TDPs of 548UAA and 548UAA PAM mutant (C770A) at 5 or 10 µM glutamate. Dashed lines represent 4 distinct FRET states. Transitions are compiled from two independent experiments. d, Dwell times of states 1-4 for 548UAA and 548UAA PAM mutant (C770A) in the presence of 5 or 10 µM glutamate. Average dwell time was calculated by fitting a single exponential decay function to dwell-time histograms for each condition. Dwell times of states 1 and 2 represent the mean of N=2 independent experiments with 86, 83, 91 and 72 total molecules examined for WT (5 µM), WT (10 µM), C770A (5 µM) and C770A (10 µM), respectively. Dwell times of states 3 and 4 represent the mean ± s.e.m. of N=3 independent experiments with 147, 140, 128 and 142 total molecules examined for WT (5 µM), WT (10 µM), C770A (5 µM) and C770A (10 µM), respectively. Transition and dwell-time analysis were performed on 100 ms data.
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Liauw, B.WH., Afsari, H.S. & Vafabakhsh, R. Conformational rearrangement during activation of a metabotropic glutamate receptor. Nat Chem Biol 17, 291–297 (2021). https://doi.org/10.1038/s41589-020-00702-5
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