Conformational fingerprinting of allosteric modulators in metabotropic glutamate receptor 2

  1. Brandon Wey-Hung Liauw
  2. Arash Foroutan
  3. Michael R Schamber
  4. Weifeng Lu
  5. Hamid Samareh Afsari  Is a corresponding author
  6. Reza Vafabakhsh  Is a corresponding author
  1. Department of Molecular Biosciences, Northwestern University, United States
4 figures, 4 tables and 1 additional file

Figures

Figure 1 with 3 supplements
Agonist-induced structural change measured at each domain using conformational fluorescence resonance energy transfer (FRET) sensors.

(A) Full-length cryo-EM structures of inactive (7EPA) and fully active (7E9G) metabotropic glutamate receptor 2 (mGluR2; human) and schematic illustrating fluorophore placement for each inter-domain sensor. (B) Representative normalized live-cell FRET trace from glutamate titration experiment on HEK293T cells expressing azi-extracellular loop 2 (azi-ECL2). Data was acquired at 4.5 s time resolution. Dose-response curves from live-cell FRET orthosteric agonist titration experiments using (C) azi-ECL2, (D) N-terminal SNAP-tag labeled mGluR2 (SNAP-m2), and (E) azi-cysteine-rich domain (azi-CRD). Data is acquired from individual cells and normalized to 1 mM glutamate response. Data represents mean ± SEM of responses from individual cells from at least three independent experiments. Total number of cells examined, mean half-maximum effective concentration (EC50), mean max response, and errors are listed in Tables 1–2.

Figure 1—figure supplement 1
Representative images and fluorescence resonance energy transfer (FRET) traces from live-cell FRET experiments.

(A) Representative image of HEK293T cells expressing N-terminal SNAP-tag labeled metabotropic glutamate receptor 2 (SNAP-m2), azi-cysteine-rich domain (azi-CRD), or azi-extracellular loop 2 (azi-ECL2) labeled with donor (left) and acceptor (right) fluorophores used for live-cell FRET experiments. Scale bar, 10 μM. (B) Representative normalized live-cell FRET traces of DCG-IV, LY379268, and (2R,4R)-APDC titration experiments on HEK293T cells expressing azi-ECL2. Data was acquired at 4.5 s time resolution.

Figure 1—figure supplement 2
Quantification of orthosteric agonist efficacy.

(A) Normalized maximal agonist-induced fluorescence resonance energy transfer (FRET) change for metabotropic glutamate receptor 2 (mGluR2) N-terminal SNAP-tag (SNAP-m2), azi-cysteine-rich domain (azi-CRD), and azi-extracellular loop 2 (azi-ECL2) sensors. Data represents mean ± SEM of responses from individual cells from at least three independent experiments. Total number of cells examined for normalization experiments, mean max response, and errors are listed in Table 2. (B) Representative normalized live-cell FRET traces from DCG-IV, LY379268, and (2R,4R)-APDC normalization experiments of azi-ECL2. Data is normalized to 1 mM glutamate response and collected at 4.5 s time resolution.

Figure 1—figure supplement 3
Orthosteric agonists examined by functional calcium imaging.

(A) Dose-response curves for metabotropic glutamate receptor 2 (mGluR2)-induced calcium flux during orthosteric agonist titrations. (B) Normalized maximal agonist-induced intracellular calcium levels. Glutamate dose-response curves for calcium flux induced by (C) azi-cysteine-rich domain (azi-CRD) and (D) azi-extracellular loop 2 (azi-ECL2). Data is normalized to 1 mM glutamate response. Data represents mean ± SEM of results from three independent experiments.

Figure 2 with 5 supplements
Positive and negative allosteric modulation of metabotropic glutamate receptor 2 (mGluR2) structural domains.

N-terminal SNAP-tag labeled mGluR2; hereafter (SNAP-m2) glutamate dose-response curves in the presence of (A) positive allosteric modulators (PAMs) or (B) NAMs. (C) Changes in glutamate potency and efficacy for SNAP-m2. The azi-cysteine-rich domain (azi-CRD) glutamate dose-response curves in the presence of (D) PAMs or (E) NAMs. (F) Changes in glutamate potency and efficacy for azi-CRD. The azi-extracellular loop 2 (azi-ECL2) glutamate dose-response curves in the presence of (G) PAMs or (H) NAMs. (I) Changes in glutamate potency and efficacy for azi-ECL2. (J) Changes in glutamate efficacy in response to PAMs and NAMs as measured by each conformational sensor. ΔPotency defined as (([modulator + glutamate]EC50 – [glutamate] EC50)/[glutamate] EC50) × 100. ΔEfficacy defined as ([1 mM glutamate + modulator] – [1 mM glutamate]) × 100. Data is acquired from individual cells and normalized to 1 mM glutamate response. Data represents mean ± SEM of responses from individual cells from at least three independent experiments. Total number of cells examined for titration and normalization experiments, mean half-maximum effective concentration (EC50), mean max response, and errors are listed in Tables 1–2.

Figure 2—figure supplement 1
Max normalization of Δ fluorescence resonance energy transfer (ΔFRET) for N-terminal SNAP-tag labeled metabotropic glutamate receptor 2 (SNAP-m2).

(A–E) Representative normalized live-cell FRET traces of SNAP-m2 normalization experiments for all positive and negative allosteric modulators tested. Data was acquired at 4 s time resolution.

Figure 2—figure supplement 2
Max normalization of Δfluorescence resonance energy transfer (ΔFRET) for azi-cysteine-rich domain (azi-CRD).

(A–E) Representative normalized live-cell FRET traces of azi-CRD normalization experiments for all positive and negative allosteric modulators tested. Data was acquired at 4.5 s time resolution.

Figure 2—figure supplement 3
Max normalization of Δfluorescence resonance energy transfer (ΔFRET) for azi-ECL2.

(A–E) Representative normalized live-cell FRET traces of azi-ECL2 normalization experiments for all positive and negative allosteric modulators tested. Data was acquired at 4.5 s time resolution.

Figure 2—figure supplement 4
Allosteric modulators examined by functional calcium imaging.

(A) Glutamate dose-response curves with and without allosteric modulators for metabotropic glutamate receptor 2 (mGluR2)-induced calcium flux. (B) Changes in glutamate potency and efficacy in response to allosteric modulator treatment, measured by intracellular calcium levels. ΔPotency defined as (([modulator + glutamate]EC50 – [glutamate] EC50)/[glutamate] EC50) × 100. ΔEfficacy defined as ([1 mM glutamate + modulator] – [1 mM glutamate]) × 100. Data is normalized to 1 mM glutamate response. Data represents mean ± SEM of results from three independent experiments.

Figure 2—figure supplement 5
Structural representation of allosteric modulator binding pocket.

The 7 transmembrane (7TM) domain (white) is from positive allosteric modulator (PAM) bound subunit of metabotropic glutamate receptor 2 (mGluR2; PDB:7MTS). Lateral view (left) and top view (right). Residues found to interact with PAM in structure (PDB: 7MTS) and from mutagenesis studies are shown with surface representations (gray). Ligands bound are superimposed volumes of PAMs (green; PDB: 7MTR, 7MTS, 7E9G) and NAMs (pink; PDB: 7EPE, 7EPF) solved in complex with metabotropic glutamate receptor 2 (mGluR2).

Figure 3 with 2 supplements
Single-molecule fluorescence resonance energy transfer (smFRET) analysis of BINA effects on cysteine-rich domain (CRD) conformational dynamics.

(A) Schematic of SiMPull assay (left) and representative image of donor and acceptor channels during data acquisition (right). Green circles indicate molecules selected by software for analysis. Scale bar, 3 μm. smFRET population histograms of azi-CRD in the presence of 0 μM, 15 μM, and 1 mM glutamate without (B–D) or with (E–G) 10 μM BINA. Histograms were fitted (black) to four Gaussian distributions centered around 0.24 (inactive; purple), 0.38 (intermediate 1; blue), 0.70 (intermediate 2; cyan), and 0.87 (active; red) FRET. Error bars represent SEM. Histograms (B–G) were generated from 332, 366, 253, 252, 418, and 367 individual particles, respectively. (H) Mean occupancy of four conformational states of azi-CRD in varying ligand conditions. Values represent area under each FRET peak from smFRET histogram as a fraction of total area. Mean and SEM values are reported in Table 3. (I) Mean cross-correlation of donor and acceptor intensities in the presence of intermediate (15 μM) and saturating (1 mM) glutamate with and without 10 μM BINA. Data was acquired at 50 ms time resolution. All data represents mean from three independent experiments.

Figure 3—figure supplement 1
Representative single-molecule fluorescence resonance energy transfer (smFRET) traces for modulator-free conditions.

(A–C) Representative smFRET traces of azi-cysteine-rich domain (azi-CRD) in the presence of (A) 0 μM, (B) 15 μM, and (C) 1 mM glutamate showing donor (green) and acceptor (red) and corresponding FRET (blue). Dashed lines represent four distinct FRET states. Data was acquired at 50 ms time resolution.

Figure 3—figure supplement 2
Representative single-molecule fluorescence resonance energy transfer (smFRET) traces for 10 μM BINA conditions.

(A–C) Representative smFRET traces of azi-cysteine-rich domain (azi-CRD) in the presence of 10 μM BINA and (A) 0 μM, (B) 15 μM, and (C) 1 mM glutamate showing donor (green) and acceptor (red) and corresponding FRET (blue). Dashed lines represent four distinct FRET states. Data was acquired at 50 ms time resolution.

Figure 4 with 2 supplements
Single-molecule fluorescence resonance energy transfer (smFRET) analysis of MNI-137 effects on cysteine-rich domain (CRD) conformational dynamics.

(A–C) smFRET population histograms of azi-CRD sensor in the presence of 0 μM (372 particles), 15 μM (560 particles), and 1 mM (479 particles) glutamate and 5 μM MNI-137. Histograms were fitted (black) to four Gaussian distributions centered around 0.24 (inactive; purple), 0.38 (intermediate 1; blue), 0.70 (intermediate 2; cyan), and 0.87 (active; red) FRET. Error bars represent SEM. (D) Mean occupancy of four conformational states of azi-CRD in varying ligand conditions. Values represent area under each FRET peak from smFRET histogram as a fraction of total area. Mean and SEM values are reported in Table 3. (E) Transition density plots of azi-CRD at 1 mM glutamate with and without MNI-137. Dashed lines represent four distinct FRET states. (F) Mean cross-correlation of donor and acceptor intensities in the presence of 0 μM, 15 μM, and 1 mM glutamate and 5 μM MNI-137. Data was acquired at 50 ms time resolution. Data represents mean from three independent experiments.

Figure 4—figure supplement 1
Representative single-molecule fluorescence resonance energy transfer (smFRET) traces for 5 μM MNI-137 conditions.

(A–C) Representative smFRET traces of azi-cysteine-rich domain (azi-CRD) in the presence of 5 μM MNI-137 and (A) 0 μM, (B) 15 μM, and (C) 1 mM glutamate showing donor (green) and acceptor (red) and corresponding FRET (blue). Dashed lines represent four distinct FRET states. Data was acquired at 50 ms time resolution.

Figure 4—figure supplement 2
Allosteric modulator effects on azi-cysteine-rich domain (azi-CRD) cross correlation.

(A) Cross-correlation of azi-CRD donor and acceptor intensities in the presence of 0 μM glutamate alone and with 5 μM MNI-137 or 10 μM BINA. (B) Cross-correlation of azi-CRD donor and acceptor intensities in the presence of 1 mM glutamate alone and with 5 μM MNI-137 or 10 μM BINA. Data was acquired at 50 ms time resolution.

Tables

Table 1
Live-cell fluorescence resonance energy transfer (FRET) titration experiment data and statistics.
SensorLigandNMean half-maximum effective concentration (EC50)SEMHill slopeStandard error
SNAP-m2Glutamate911.91.5–1.440.08
SNAP-m2DCG-IV60.40.1–1.260.11
SNAP-m2LY379268630.69.3–1.120.07
SNAP-m2(2R,4R)-APDC66.93.1–1.100.05
SNAP-m2Glutamate + 10 μM BINA231.20.4–1.240.09
SNAP-m2Glutamate + 5 μM LY48737943.80.9–1.430.11
SNAP-m2Glutamate + 0.5 μM JNJ-4215360554.21.9–0.950.05
SNAP-m2Glutamate + 10 μM MNI-137417.22.8–1.610.06
SNAP-m2Glutamate + 10 μM Ro 64–5229319.62.6–1.520.04
azi-CRDGlutamate2611.60.51.190.03
azi-CRDDCG-IV101.10.20.940.10
azi-CRDLY3792682012.10.51.360.05
azi-CRD(2R,4R)-APDC366.51.21.100.05
azi-CRDGlutamate + 10 μM BINA101.60.31.160.05
azi-CRDGlutamate + 5 μM LY487379224.50.60.910.04
azi-CRDGlutamate + 0.5 μM JNJ-42153605104.71.30.840.03
azi-CRDGlutamate + 10 μM MNI-1372713.80.71.100.04
azi-CRDGlutamate + 10 μM Ro 64–52291316.91.21.050.06
azi-ECL2Glutamate155.10.60.960.07
azi-ECL2DCG-IV240.90.11.050.06
azi-ECL2LY379268910.22.41.030.04
azi-ECL2(2R,4R)-APDC136.71.31.140.05
azi-ECL2Glutamate + 10 μM BINA162.50.21.060.07
azi-ECL2Glutamate + 5 μM LY487379223.50.20.980.05
azi-ECL2Glutamate + 0.5 μM JNJ-42153605172.20.10.970.06
azi-ECL2Glutamate + 10 μM MNI-137814.41.71.320.06
azi-ECL2Glutamate + 10 μM Ro 64–5229517.42.41.070.09
  1. All EC50 and errors values are in μM, except for LY379268 (nM).

Table 2
Live-cell fluorescence resonance energy transfer (FRET) max normalization experiment data and statistics.
SensorLigandNMean max responseSEM
SNAP-m2Glutamate-1-
SNAP-m2DCG-IV250.790.01
SNAP-m2LY379268231.010.01
SNAP-m2(2R,4R)-APDC140.960.01
SNAP-m2Glutamate + 10 μM BINA71.020.01
SNAP-m2Glutamate + 5 μM LY487379141.070.01
SNAP-m2Glutamate + 0.5 μM JNJ-42153605221.010.01
SNAP-m2Glutamate + 10 μM MNI-137220.850.02
SNAP-m2Glutamate + 10 μM Ro 64–5229350.870.01
azi-CRDGlutamate-1-
azi-CRDDCG-IV190.690.01
azi-CRDLY379268251.060.02
azi-CRD(2R,4R)-APDC131.020.01
azi-CRDGlutamate + 10 μM BINA91.120.05
azi-CRDGlutamate + 5 μM LY487379191.560.07
azi-CRDGlutamate + 0.5 μM JNJ-4215360581.430.08
azi-CRDGlutamate + 10 μM MNI-137180.860.02
azi-CRDGlutamate + 10 μM Ro 64–5229180.590.03
azi-ECL2Glutamate-1-
azi-ECL2DCG-IV250.640.02
azi-ECL2LY379268221.140.04
azi-ECL2(2R,4R)-APDC561.050.01
azi-ECL2Glutamate + 10 μM BINA141.420.07
azi-ECL2Glutamate + 5 μM LY48737971.250.09
azi-ECL2Glutamate + 0.5 μM JNJ-42153605130.990.02
azi-ECL2Glutamate + 10 μM MNI-137580.780.03
azi-ECL2Glutamate + 10 μM Ro 64–522980.840.05
  1. All max response values are normalized to 1 mM glutamate.

Table 3
Single-molecule fluorescence resonance energy transfer (smFRET) state occupancy data and statistics.
ModulatorGlut (μM)State (#)Mean occupancySEM
None010.360670.048
None020.565260.02692
None030.066150.02385
None040.007920.00792
None1510.019320.01049
None1520.276990.06688
None1530.298990.01579
None1540.40470.09147
None100010.006420.00292
None100020.078410.01209
None100030.311310.02404
None100040.603860.01743
10 μM BINA010.305270.02468
10 μM BINA020.519940.04492
10 μM BINA030.148260.04699
10 μM BINA040.026530.01748
10 μM BINA1510.014240.00761
10 μM BINA1520.112170.01526
10 μM BINA1530.33670.07918
10 μM BINA1540.536880.07621
10 μM BINA100010.002960.00154
10 μM BINA100020.037510.00782
10 μM BINA100030.217910.01663
10 μM BINA100040.741620.01093
5 μM MNI-137010.748610.02014
5 μM MNI-137020.221980.01316
5 μM MNI-137030.020380.01004
5 μM MNI-137040.009030.00541
5 μM MNI-1371510.103870.02484
5 μM MNI-1371520.749370.01688
5 μM MNI-1371530.127240.01026
5 μM MNI-1371540.019520.00254
5 μM MNI-137100010.002070.000954
5 μM MNI-137100020.55970.02561
5 μM MNI-137100030.330980.03204
5 μM MNI-137100040.107250.00734
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (Homo sapiens)HEK 293TSigma AldrichCat # 12022001
Transfected construct (Mus musculus)SNAP-m2Liauw et al., 2021
Transfected construct (Mus musculus)SNAP-m2 (no-FLAG)Liauw et al., 2021 (modified)
Transfected construct (Mus musculus)azi-CRDLiauw et al., 2021
Transfected construct (Mus musculus)azi-ECL2Genscript (modified)ORF clone: OMu19627D
Transfected construct (Homo sapiens)pIRE4-AziAddgenePlasmid # 105,829
Transfected construct (Mus musculus)Gqo5Addgene (modified)Plasmid # 24,500
Chemical compound, drugGlutamateSigma AldrichCat # 6106-04-3
Chemical compound, drugLY379268TocrisCat # 2,453
Chemical compound, drugDCG-IVTocrisCat # 0975
Chemical compound, drug(2R,4R)-APDCTocrisCat # 1,208
Chemical compound, drugLY487379TocrisCat # 3,283
Chemical compound, drugBINATocrisCat # 4,048
Chemical compound, drugJNJ-42153605Cayman Chemical21,984
Chemical compound, drugRo 64–5229TocrisCat # 2,913
Chemical compound, drugMNI-137TocrisCat # 4,388
Chemical compound, drugSNAP-Surface Alexa Fluor 549New England BiolabsS9112S
Chemical compound, drugSNAP-Surface Alexa Fluor 647New England BiolabsS9136S
Chemical compound, drugOregon Green 488 BAPTA-1, AMThermo Fisher ScientificO6807
Chemical compound, drugCy3 AlkyneClick Chemistry ToolsTA117-5
Chemical compound, drugCy5 AlkyneClick Chemistry ToolsTA116-5
Chemical compound, drug4-azido-L-phenylalanineChem-Impex InternationalCat # 06162
Chemical compound, drugAminoguanidine (hydrochloride)Cayman Chemical81,530
Chemical compound, drugBTTESClick Chemistry Tools1237–500
Chemical compound, drugCopper (II) sulfateSigma AldrichCat # 451657–10 G
Chemical compound, drug(+)-Sodium L-AscorbateSigma AldrichCat # 11140–250 G
Chemical compound, drugGlutamic-Pyruvic TransaminaseSigma AldrichCat # G8255-200UN
Chemical compound, drugSodium PyruvateGibco11360–070
Chemical compound, drugDMEMCorning10–013-CV
Chemical compound, drugDefined Fetal Bovine SerumThermo Fisher ScientificSH30070.03
Chemical compound, drugPenicillin-StreptomycinGibco15140–122
Chemical compound, drugLipofectamine 3000 Transfection ReagentThermo Fisher ScientificL3000015
Chemical compound, drugPoly-L-lysine hydrobromideSigma AldrichCat # P2636
Chemical compound, drugFLAG-tag antibodyGenscriptA01429
Software, algorithmsmCamera (Version 1.0)http://ha.med.jhmi.edu/resources/
Software, algorithmImageJ (Version 1.52 p)http://imagej.nih.gov/ij/RRID:SCR_003070
Software, algorithmOriginPro (2020b)https://www.originlab.com/RRID:SCR_014212
Software, algorithmAdobe Illustrator (2022)https://www.adobe.com/RRID:SCR_010279

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  1. Brandon Wey-Hung Liauw
  2. Arash Foroutan
  3. Michael R Schamber
  4. Weifeng Lu
  5. Hamid Samareh Afsari
  6. Reza Vafabakhsh
(2022)
Conformational fingerprinting of allosteric modulators in metabotropic glutamate receptor 2
eLife 11:e78982.
https://doi.org/10.7554/eLife.78982