Abstract
Metabotropic glutamate receptors belong to a family of G protein-coupled receptors that are obligate dimers and possess a large extracellular ligand-binding domain that is linked via a cysteine-rich domain to their 7-transmembrane domain1. Upon activation, these receptors undergo a large conformational change to transmit the ligand binding signal from the extracellular ligand-binding domain to the G protein-coupling 7-transmembrane domain2. In this manuscript, we propose a model for a sequential, multistep activation mechanism of metabotropic glutamate receptor subtype 5. We present a series of structures in lipid nanodiscs, from inactive to fully active, including agonist-bound intermediate states. Further, using bulk and single-molecule fluorescence imaging, we reveal distinct receptor conformations upon allosteric modulator and G protein binding.
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Data availability
The data that support this study are available from the corresponding authors upon request. The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession codes EMD-41092, EMD-41099, EMD-41139 and EMD-41069. Model coordinates have been deposited in the Protein Data Bank (PDB) under accession numbers 8T7H, 8T8M, 8TAO and 8T6J. Previously published structures can be accessed via accession codes: 6N50, 6N51, 6N52, 7FD9, 6N4Y, 6FFI, 7MTR, 7MTS. EMBD codes of available structures mentioned in this manuscript: EMD-0345 or EMD-0346. Source data are provided with this paper.
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Acknowledgements
We thank D. Hilger for helpful discussions. The cryo-EM data were collected at Stanford Cryo-Electron Microscopy Center (cEMc). This work was supported by National Institutes of Health grants no. K99GM148823 (N.R.L.), no. R01NS119826 (E.Y.I.) and no. R01NS028471 (B.K.K.). We thank the National Institute of Drug Abuse (NIDA). E.Y.I. is a Weill Neurohub Investigator. S.M. and B.K.K. are Chan Zuckerberg Biohub Investigators.
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Contributions
K.K.K. and B.K.K. conceived the project. K.K.K. prepared samples, froze grids and collected cryo-EM data with help from C.Z. and E.M. K.K.K. and J.X. developed the minimal cysteine mGlu5 construct and performed bimane studies, and made smFRET samples with help from E.S.O. H.W. processed cryo-EM data, performed the 3DVA and 3DFlex analysis and performed molecular dynamics simulations. C.H. collected and analysed smFRET data under the supervision of E.Y.I. N.R.L. collected and analysed the HDX-MS data under the supervision of S.M. A.K. helped with structure analysis. K.K.K., H.W. and B.K.K. wrote the manuscript with input from all authors.
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B.K.K. is co-founder of and consultant for ConfometRx. The remaining authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 mGlu5 activation in detergent compared to lipid environment.
(a) Structural domains of mGlu5. (b) Representative size exclusion chromatography trace and SDS-PAGE gel (for gel source data, see Supplementary Fig. 1a) of mGlu5 in nanodisc (n = 6). (c) GTP turnover assay showing mGlu5 induced Gq turnover. In the presence of agonist Quis (20 μM), mGlu5 in detergent does not induce significant Gq (red) turnover compared to Gq alone (grey). The addition of Quis and CDPPB (20 μM) (dark green) to mGlu5 in detergent results in a small but significant increase in G protein turnover. With mGlu5 in nanodiscs, the addition of Quis significantly increases Gq turnover (magenta). Quis and CDPPB (light green) further increase the GTP turnover of Gq. The negative allosteric modulator, MTEP inhibits turnover in mGlu5 nanodiscs condition (blue). Data represented as mean ± s.d., ns= 0.4124, p < 0.0001****, unpaired t-test (two-tailed) performed for statistical analysis between two conditions. One-way ANOVA performed for comparison across multiple conditions showed p < 0.0001. n = 7 individual experiments (data normalization was done with the average value of Quis-bound mGlu5 in nanodiscs as 100% and receptor alone as 0%). (d) In the presence of the agonist iperoxo, muscarinic acetylcholine M1 receptor (in MNG) induces significant GTP turnover in Gq (p < 0.0001****). But no difference is seen with Quis-bound mGlu5 (in MNG) and Gq (ns = 0.5374). Data represented as mean ± s.d., p values are from unpaired t-test (two-tailed) performed for statistical analysis between two conditions. One-way ANOVA performed for comparison across multiple conditions showed p < 0.0001. n = 4 individual experiments. Data normalization was done with the average value of Gq in MNG as 100% and buffer alone as 0%.
Extended Data Fig. 2 HDX-MS of mGlu5 in detergent and nanodisc.
(a) HDX-MS data are shown as Woods plots, i.e., as the difference in the percent deuteration for a given peptide at a given time point against the sequence position for Apo mGlu5 in detergent (GDN) vs 5 mM glutamate-bound mGlu5 in detergent (GDN) (top) and Apo mGlu5 in detergent (GDN) vs 5 mM glutamate-bound mGlu5 in nanodisc (HDL) (bottom). Black boxes numbered 1- 4 are example regions in the VFT that show differences in deuterium uptake between the Apo and 5 mM glutamate conditions, regardless of the membrane environment, but no detectable difference between mGlu5 in detergent (GDN) and nanodisc (HDL) in the presence of 5 mM glutamate (the corresponding deuterium uptake plots are shown on the right). The red box is a region in the TM that shows a difference between agonist-bound mGlu5 in detergent and nanodisc (HDX-MS exchange curves shown in Extended Data Fig. 2c). All measurements done in triplicates, data represented at mean ± s.e.m. Woods plots are shown for a single replicate. (b) HDX-MS changes in Apo mGlu5 in detergent and agonist-bound mGlu5 in nanodisc are plotted onto the mGlu5 structure (PDB code: 6N51). (c) The region of TM3 where peptides were observed in HDX-MS measurements is shown in red. Deuterium uptake plots of these TM3 peptides (n = 3, red box) show that receptor in GDN under the Apo condition (black) and in the presence of 5 mM glutamate (yellow) overlay well, whereas TM3 peptides of mGlu5 in nanodisc in the presence of 5 mM glutamate (magenta) exhibit reduced uptake relative to both GDN conditions. (d) Agonist-bound mGlu2 (PDB code: 7MTR) is overlayed with agonist-bound mGlu2-G protein complex (PDB code: 7MTS) showing conformational changes in the intracellular region of TM3.
Extended Data Fig. 3 Cryo-EM data processing workflow and resolution assessment of Quis-bound maps.
(a) Workflow of cryo-EM data processing to obtain Quis-bound Intermediate 1a and Quis-bound Intermediate 2a structures. Also shown is the density for Quis and residues around the ligand in the two structures. (b) Local resolution maps of the Quis-bound structures. (c) Angular particle distribution of the Quis-bound structures. (d) Gold-standard FSC curves of the structures.
Extended Data Fig. 4 Comparison of mGlu5 structures upon activation by Quis.
(a) Overlay of the Apo (grey, PDB: 6N52) and Quis-bound Intermediate 1a showing CRDs and TMDs in an “inactive” state. Insert shows the Quis binding pocket. (b) Movement of the VFTs upon agonist binding in Quis-bound Intermediate 1a state compared to the Apo state (PDB 6N52, grey). Arrows represent the movement of every 5 Cα atoms from the Apo to the Intermediate 1a state upon Quis binding. Nb43 is shown in yellow. (c) To get insights into structural changes needed to initiate activation, we compared the Quis-bound Intermediate 1a (light pink) and the antagonist, LY341495-bound (PDB: 7FD9, dark green) mGlu5 structures. LY341495 binding to the receptor inhibits the movement of residues W100 and E279. (d) Comparing the movement of the VFTs in the Quis-bound Intermediate 1a (light pink, magenta) and the Quis-bound Intermediate 2a states (cyan and teal) show large rearrangements in the lower lobe, with relatively smaller changes in the upper lobe. Arrows represent the movement of every 5 Cα atoms from the Intermediate 1a state to the Intermediate 2a state. (e) Single protomer alignment of Quis-bound Intermediate 2a (cyan) and Quis-bound Intermediate 1a (light pink) structures show no change in the Quis binding pocket. (f) Overlay of Apo (grey, PDB: 6N52) and Quis-bound Intermediate 1a states show minimal changes in the CRDs and TMs. Arrows represent the movement of every 5 Cα atoms from Apo to Intermediate 1a. (g) Large changes in the CRDs and TMs are seen when comparing the Quis-bound Intermediate 1a and the Quis-bound Intermediate 2a states. Arrows represent the movement of every 5 Cα atoms from Intermediate 1a state to Intermediate 2a. (h) The CRDs in the Quis-bound Intermediate 1a structure are separated by ~38 Å (as measured at residue E527). In the Quis-bound Intermediate 2a state, the twisting of the lower lobe enables the CRDs (~11 Å at residue E527) and TMs to move adjacent to each other. (i) The TMs in the Quis-bound Intermediate 1a structure are far apart with TM5 being the most proximal helix pair (~21 Å). In the Quis-bound Intermediate 2a state the TMs of the protomers, in addition to moving closer to each other, rotate ~20o to form a TM6-TM6 interface, a hallmark of Family C activation.
Extended Data Fig. 5 Cryo-EM data processing workflow and resolution assessment of CDPPB, Quis-bound map.
(a) Workflow of cryo-EM data processing to obtain CDPPB, Quis-bound mGlu5 structure, Intermediate 3a. Also shown is the density for Quis and CDPPB, with density also shown for residues around the ligands. (b) Local resolution maps of the CDPPB, Quis-bound mGlu5 structure. (c) Angular particle distribution of the structure. (d) Gold-standard FSC curves of the Quis-bound mGlu5 structure.
Extended Data Fig. 6 Cryo-EM data processing workflow and resolution assessment of CDPPB-bound map.
(a) Workflow of cryo-EM data processing to obtain CDPPB-bound mGlu5 Intermediate 1b structure. The density of CDPPB and the residues around the ligand are shown. (b) Local resolution maps of the CDPPB-bound mGlu5 structure. (c) Angular particle distribution of the structure. (d) Gold-standard FSC curves of the CDPPB-bound mGlu5 structure.
Extended Data Fig. 7 CDPPB-bound structure analysis.
(a) Cryo-EM density and model of CDPPB-bound mGlu5 Intermediate 1b in a nanodisc. Also shown is the density for the two bound CDPPB, one in each TM domain. (b) Comparison of the allosteric binding pocket in Apo (PDB:6N52, grey) and CDPPB-bound mGlu5 (dark blue), shows changes in TM5 (N7475.47) and TM6 (W7856.50) to accommodate CDPPB (slate). (c) Overlay of CDPPB from Intermediate 1b (dark blue) and Intermediate 3a structures showing minimal changes in the conformation of TM5 and TM6. (d) Residues that interact with CDPPB only in Intermediate 3a are shown in green (T7816.46 and C7826.47) and those that interact with CDPPB only in Intermediate 1b are shown in blue (I7515.51).
Extended Data Fig. 8 CDPPB, Quis-bound structural analysis.
(a) Overlay of intersubunit B and C helices in Quis-bound Intermediate 2a state and CDPPB, Quis-bound Intermediate 3a structure. Residues R114 and E111 interact in both structures. (b) Overlay of Quis binding pocket in Quis-bound Intermediate 2a and CDPPB, Quis-bound Intermediate 3a structures, showing no difference in the ligand pocket. (c) The conformation of residue W7856.50 is different in the structure with the NAM, MPEP (PDB: 6FFI, brown) compared to that with the PAM, CDPPB (dark green). (d) TM6 in the CDPPB-bound Protomer 1 has moved outward compared to Protomer 2 with no CDPPB bound. In CDPPB-bound Protomer 1, Y7796.44 points towards the intersubunit interface, as seen in (e). Though we cannot model the Y7796.44 sidechain in Protomer 1 with confidence due to a lack of good density, we have added the most frequently occurring rotomer of Tyr. (f) Comparison of the allosteric pocket in CDPPB-bound protomer (protomer 1, dark green and CDPPB shown as orange) and the protomer with no CDPPB (protomer 2, green).
Extended Data Fig. 9 Molecular Dynamics (MD) simulations of CDPPB.
(a) Three replicates of the MD trajectories showing the RMSD of mGlu5 (blue), of the residue W7856.50 (red) and CDPPB (green). Data represented as mean ± s.e.m. (b) The Starting model and the MD trajectory superpositions are shown. (c) Snapshots of the CDPPB and W7856.50 every 100 ns is shown.
Extended Data Fig. 10 Characterisation of minimal cysteine mGlu5 and ICL2 conformation.
(a) Residues Cys6914.30 and Cys681ICL2 that contribute to background labeling with dyes are shown as spheres. Other cysteine residues in the receptor are shown as yellow sticks. (b) mGlu5 full-length and ECD alone (VFT and CRD) were labeled with the cysteine reactive dye, monobromobimane. Though no signal was seen for ECD (dark grey), full-length (FL) mGlu5 produced a bimane spectrum (light grey). This implies that mGlu5 TMs have cysteine residues that are exposed to being labeled with bimane. n = 1 individual experiment. (c) WT and minimal cysteine (C6914.30A and C681ICL2A) constructs were labeled with Atto488. Unlike WT, the minimal cysteine construct exhibits almost no background labeling for the times tested (for gel source data, see Supplementary Fig. 1b). (d) Fluorescence intensity at 464 nm for mGlu5 WT labeled with bimane (reading out on ICL2 conformation from Fig. 3d) is plotted for the different ligand conditions. Though there is no significant difference between Apo (light grey) and Quis (cyan), the addition of Quis and CDPPB (dark green) showed a significant change. No further change was detected with the addition of Gq to the Quis and CDPPB condition (yellow). The addition of MTEP resulted in a significant decrease in fluorescence intensity (dark grey). Data represented as mean ± s.d., ns = 0.5326, p = 0.0001***, p = 0.0026**, p < 0.0001****, unpaired t-test (two-tailed) performed for statistical analysis between two conditions. One-way ANOVA performed for comparison across multiple conditions showed p < 0.0001, n = 3 individual experiments. (e) Bimane spectra of mGlu5 in nanodiscs labeled only at C681ICL2 (C6914.30A construct). Unlike adding Quis (cyan) which resulted in no change in the spectrum, the addition of CDPPB alone (blue) or Quis and CDPPB (dark green) increases the fluorescence. On the other hand, LY341495 and MTEP (brown) cause a decrease in fluorescence. Data represented as mean ± s.e.m., n = 3 individual experiments. (f) Plotting the fluorescence intensity at 464 nm for bimane data in Extended Data Fig. 10e shows a significant difference between CDPPB alone (blue), Quis and CDPPB (dark green), and LY341495 and MTEP (brown) compared to Apo (grey). Data represented as mean ± s.e.m., ns = 0.5713, p < 0.0001, p = 0.0257* (Apo vs CDPPB), p < 0.0160* (Apo vs Quis + CDPPB), unpaired t-test (two-tailed) performed for statistical analysis between two conditions. One-way ANOVA performed for comparison across multiple conditions showed p < 0.0001, n = 3 individual experiments. (g) Comparison of Quis-bound (cyan) and CDPPB, Quis-bound structures (dark green) showing changes in TM3 and TM4 (~2.6 Å). Also shown is the position of residue C6914.30 which is bimane labeled in the WT construct (Fig. 3d, Extended Data Fig. 10e).
Extended Data Fig. 11 Fractional occupancy determined from FRET histograms with three component Gaussian fits.
(a) – (i) FRET histogram with three component Gaussian fits for each ligand condition. (j) Percentage area under the peak and peak center values. Apo (n = 319), Quis (n = 392), Quis + CDPPB (n = 329), Quis + Gq (n = 306), Quis + CDPPB + Gq (n = 317), LY341495 (LY) (n = 245), CDPPB (n = 329) and CDPPB + Gq (n = 347). Data represented as mean ± s.e.m.
Extended Data Fig. 12 smFRET fitting statistics and analysis.
(a) Plot of the Akaike information criterion (corrected for small sample size, AICc) values for analysis with 1 to 5 Gaussians fits for the smFRET data. The AICc values showed broad minima at 3 and 4 fits. Three Gaussians were used to fit the data. (b) smFRET data showing the comparison of Apo (grey, n = 319) and antagonist-bound mGlu5 (brown, n = 245). Data represented as mean ± s.e.m. (c) The addition of CDPPB alone results in two FRET peaks, one at 0.25, Intermediate 1b state, and the other at 0.6, the Intermediate 2b state (slate, n = 329). In the presence of Quis (teal), the same two FRET peaks are seen except with different relative proportions of the two states. The addition of Gq to the Quis-alone sample shifts the population to the high FRET states, Intermediate 3b (0.75) and Fully Active (0.9) at the expense of the Intermediate 2a (0.6) and Intermediate 1a (0.25) peaks. For the CDPPB alone sample, the addition of Gq results in the appearance of a high FRET peak with a decrease, but not complete disappearance of the Intermediate 2b (0.6) and Intermediate 1b (0.25) peaks (n = 347). Data represented as mean ± s.e.m. (d) Example smFRET traces showing donor (green), and acceptor (red) intensity values as well as the calculated FRET values (blue) for a series of ligand conditions with and without Gq.
Extended Data Fig. 13 Transition Density plots for different ligand conditions.
(a) In the Apo state, we see transitions between the 0.25 and 0.4 FRET states. (b) With the addition of Gq, we see higher FRET state transitions, in addition to the 0.25 to 0.4 transitions. (c) In the presence of Quis, we see transitions between 0.4 and 0.6 FRET states. (d) Adding Gq to Quis-bound mGlu5 results in transitions between 0.6 and 0.75 states and between 0.75 and 0.9 states. No transition is seen between 0.6 and 0.9 states, indicating a stepwise transition from 0.6 to 0.75 to 0.9 states. (e) With Quis and CDPPB, transitions are seen between 0.4 and 0.6 and between 0.6 and 0.75 FRET states. Also, there are off-diagonal transitions between 0.4 and 0.75 states, indicating that CDPPB lowers the energy of the 0.75 state so that the 0.4 to 0.75 transitions can occur. This 0.4 to 0.75 transition is not seen with other ligand conditions. (f) Adding Gq to Quis and CDPPB results in more 0.6 to 0.75 transitions and the appearance of the 0.75 to 0.9 transitions. (g) CDPPB alone results in mostly transitions between 0.25 and 0.4, 0.4 and 0.6 and 0.6 and 0.75 FRET states. (h) Adding Gq to CDPPB results in higher FRET transitions, in addition to 0.4 to 0.6 and 0.6 to 0.75 transitions. The transition of 0.25 to 0.4 FRET state is minimal. (i) In LY341495, we see low FRET state transitions, indicating stabilization of inactive states.
Supplementary information
Supplementary Information
This file contains Supplementary Figs. 1 and 2 and Table 1.
Supplementary Video 1
3D variability analysis (3DVA) of Quis-bound intermediate 2a and of Quis, CDPPB-bound intermediate 2a. The 3DVA analysis performed on the cryo-EM data of Quis-bound intermediate 2a and of Quis, CDPPB-bound intermediate 3a showing a ‘stretch’ and a ‘swing’ motion between the VFT and TM, and a nanodisc diameter variability.
Supplementary Video 2
3DFlex refinement of Quis, CDPPB-bound intermediate 2a. The 3Flex refinement performed on the cryo-EM data of Quis, CDPPB-bound intermediate 3a showing a ‘swing’, ‘asymmetric stretch’ and ‘squeeze’ motions.
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Krishna Kumar, K., Wang, H., Habrian, C. et al. Stepwise activation of a metabotropic glutamate receptor. Nature (2024). https://doi.org/10.1038/s41586-024-07327-x
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DOI: https://doi.org/10.1038/s41586-024-07327-x
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