1. Structural Biology and Molecular Biophysics
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Molecular mechanism of TRPV2 channel modulation by cannabidiol

  1. Ruth A Pumroy
  2. Amrita Samanta
  3. Yuhang Liu
  4. Taylor ET Hughes
  5. Siyuan Zhao
  6. Yevgen Yudin
  7. Tibor Rohacs
  8. Seungil Han  Is a corresponding author
  9. Vera Y Moiseenkova-Bell  Is a corresponding author
  1. Perelman School of Medicine, University of Pennsylvania, United States
  2. Pfizer Research and Development, United States
  3. New Jersey Medical School, Rutgers University, United States
Research Article
Cite this article as: eLife 2019;8:e48792 doi: 10.7554/eLife.48792
4 figures, 1 table, 8 data sets and 1 additional file

Figures

Figure 1 with 10 supplements
Overview of the full-length rat TRPV2 channel structures in nanodiscs.

(A) Three-dimensional cryo-EM reconstructions for TRPV2APO_1 at 3.7 Å (salmon), TRPV2APO_2 at 4.0 Å (purple), TRPV2CBD_1 at 3.4 Å (green), TRPV2CBD_2 at 3.2 Å (blue); (B) CBD binding pocket in the TRPV2CBD_2 structure. The S5 and S6 helices are shown in blue and gray, respectively. Density attributed to CBD is shown in pink. (C) The profile of the ion permeation pathway for TRPV2APO_1 (salmon), TRPV2APO_2 (purple), TRPV2CBD_1 (green) and TRPV2CBD_2 (blue) structures; (D) Graphical representation of the radius of the pore as a function of the distance along the ion conduction pathway.

https://doi.org/10.7554/eLife.48792.002
Figure 1—figure supplement 1
EM summary of apo full-length rat TRPV2 in nanodiscs.

(A) Representative micrograph and 2D classes of cryo-EM data. Map FSC curves and model validation curves for (B) TRPV2APO_1 and (C) TRPV2APO_2. Angular distribution for the final 3D reconstruction of (D) TRPV2APO_1 and (F) TRPV2APO_2. Tall red cylinders indicate a large number of particles aligned at a position and short blue cylinders indicate fewer particles. (D) Side and bottom views of (E) TRPV2APO_1 and (G) TRPV2APO_2 colored based on local resolution as calculated via RELION. Blue, white and red regions indicate resolutions of 3.5 Å, 4.5 Å and 5.5 Å, respectively.

https://doi.org/10.7554/eLife.48792.003
Figure 1—figure supplement 2
Apo TRPV2 cryo-EM data processing.

Workflow for 3D reconstruction resulting in two distinct apo TRPV2 structures.

https://doi.org/10.7554/eLife.48792.004
Figure 1—figure supplement 3
EM summary of CBD-bound full-length rat TRPV2 in nanodiscs.

(A) Representative micrograph and 2D classes of cryo-EM data. Map FSC curves and model validation curves for (B) TRPV2CBD_1 and (C) TRPV2CBD_2. Angular distribution for the final 3D reconstruction of (D) TRPV2CBD_1 and (F) TRPV2CBD_2. Tall red cylinders indicate a large number of particles aligned at a position and short blue cylinders indicate fewer particles. (D) Side and bottom views of (E) TRPV2CBD_1 and (G) TRPV2CBD_2 colored based on local resolution as calculated via RELION. Blue, white and red regions indicate resolutions of 3.0 Å, 4.0 Å and 5.0 Å, respectively.

https://doi.org/10.7554/eLife.48792.005
Figure 1—figure supplement 4
CBD-bound TRPV2 cryo-EM data processing.

Workflow for 3D reconstruction resulting in two distinct CBD-bound TRPV2 structures.

https://doi.org/10.7554/eLife.48792.006
Figure 1—figure supplement 5
Representative densities from the TRPV2APO_1 cryo-EM map.

Densities are contoured at σ = 5.

https://doi.org/10.7554/eLife.48792.007
Figure 1—figure supplement 6
Representative densities from the TRPV2APO_2 cryo-EM map.

Densities are contoured at σ = 5.

https://doi.org/10.7554/eLife.48792.008
Figure 1—figure supplement 7
Representative densities from the TRPV2CBD_1 cryo-EM map.

Densities are contoured at σ = 5.

https://doi.org/10.7554/eLife.48792.009
Figure 1—figure supplement 8
Representative densities from the TRPV2CBD_2 cryo-EM map.

Densities are contoured at σ = 5.

https://doi.org/10.7554/eLife.48792.010
Figure 1—figure supplement 9
Apo full-length rat TRPV2 pore turret comparison.

Comparison of cryo-EM density maps of TRPV2APO_1 (left, salmon), TRPV2APO_2 (middle, purple) and full-length rat TRPV2 in detergent (EMD 7118; right, green) in the pore turret region. The density surrounding the pore helix and the top of S5 is colored to standout from the rest of the density. A cartoon model of a single monomer is shown in each map. Arrows indicate the final residues visible on each end of the pore turret.

https://doi.org/10.7554/eLife.48792.011
Figure 1—figure supplement 10
Cryo-EM density in the CBD binding pocket.

TRPV2 models overlaid with the final postprocessed map and the two corresponding half maps for (A) TRPV2APO_1, (B) TRPV2APO_2, (C) TRPV2CBD_1, and (D) TRPV2CBD_2. Cryo-EM densities are contoured at σ = 5 and are shown as gray mesh. Side chains of interest are shown as sticks. Bound CBD is not shown.

https://doi.org/10.7554/eLife.48792.012
Figure 2 with 4 supplements
The CBD binding site.

Model representations of the CBD binding pockets in the (A) TRPV2APO_1 (salmon), (B) TRPV2CBD_1 (green) and (C) TRPV2CBD_2 (blue) structures. CBD is shown as pink sticks. Residues of interest are labeled and represented as sticks.

https://doi.org/10.7554/eLife.48792.014
Figure 2—figure supplement 1
CBD fit in the cryo-EM map.

(A) Chemical diagram of cannabidiol (CBD). (B) Fit of CBD into the TRPV2CBD_1 map. (C) Comparison of two poses of CBD in the TRPV2CBD_1 map. The residue shown in sticks is Leu537. The red arrow indicates a clash. (D) Fit of CBD into the TRPV2CBD_2 map.

https://doi.org/10.7554/eLife.48792.015
Figure 2—figure supplement 2
Hydrogen binding between CBD and the helical backbone.

The CBD binding pocket of (A) TRPV2CBD_1 (green) and (B) TRPV2CBD_2 (blue) represented as cartoons and sticks. Dashed yellow lines indicate measured distances and residues of interest are labeled.

https://doi.org/10.7554/eLife.48792.016
Figure 2—figure supplement 3
Sequence alignment of thermoTRPV channels.

Alignment of human thermo TRPV channels and rat TRPV2 (highlighted in green). Residues are colored using the Clustal X coloration scheme. Residues of interest in this study are highlighted in yellow.

https://doi.org/10.7554/eLife.48792.017
Figure 2—figure supplement 4
The double mutant L541F-L631F increases current responses to CBD.

Whole cell patch clamp experiments in HEK293T cells were performed as described in the Materials and methods section. Representative current traces in response to 20 μM CBD and 100 μM 2-APB at 100 mV (upward deflections) and at −100 mV (downward deflections) in cells transfected with (A) wild type TRPV2 and with (B) Leu541Phe-Leu631Phe mutant. (C) Summary of the ratio of currents induced by CBD and 2-APB. Statistical significance was determined by the non-parametric Kolmogorov Smirnov test.

https://doi.org/10.7554/eLife.48792.018
Figure 3 with 3 supplements
Conformational changes upon CBD binding.

(A) Overlay of the TRPV2APO_1 (salmon) and TRPV2CBD_1 (green) structures, aligned to the tetrameric pore (S5–PH–S6). (B) Zoom view from the intracellular side of the membrane on the overlay between TRPV2APO_1 (salmon) and TRPV2CBD_1 (green) structures. (C) Overlay of one ARD from the TRPV2APO_1 (salmon) and TRPV2CBD_1 (green) structures with helices displayed as cylinders. (D) Overlay of the TRPV2APO_1 (salmon) and TRPV2CBD_2 (blue) structures, aligned to the tetrameric pore (S5–PH–S6). (E) Zoom view from the intracellular side of the membrane on the overlay between TRPV2APO_1 (salmon) and TRPV2CBD_2 (blue) structures. (F) Overlay of one ARD in the TRPV2APO_1 (salmon) and TRPV2CBD_2 (blue) structures with helices displayed as cylinders. Residues of interest are represented as sticks and labeled. Red arrows indicate the junction between the S4-S5 linker and S5. Dashed black lines are references to indicate rotation. The measurements for rotation and lateral shifts are labeled and indicated with black arrows.

https://doi.org/10.7554/eLife.48792.019
Figure 3—figure supplement 1
Cryo-EM map density around the S4-S5 linker, S5, and the TRP helices.

Density for (A) TRPV2APO_1 (salmon), (B) TRPV2APO_2 (purple), (C) TRPV2CBD_1 (green), and (D) TRPV2CBD_2 (blue) is contoured to σ = 5. The view of the S4-S5 linker and S5 is from a single monomer. The TRP helix tetramer is viewed from the intracellular side of the membrane.

https://doi.org/10.7554/eLife.48792.020
Figure 3—figure supplement 2
Cryo-EM maps highlighting the CTD and NTD.

Cryo-EM maps showing the overall channel in gray with the NTD and CTD highlighted. (A) Cryo-EM map of TRPV2APO_1 with density for the NTD colored salmon. (B) Cryo-EM map of TRPV2APO_2 with density for the NTD colored purple. (C) Cryo-EM map of TRPV2CBD_1 with density for the NTD colored green. (D) Cryo-EM map of TRPV2CBD_2 with density for the CTD colored blue. A red circle indicates the end of the CTD in TRPV2APO_1, TRPV2APO_2, and TRPV2CBD_1.

https://doi.org/10.7554/eLife.48792.021
Figure 3—figure supplement 3
Cryo-EM map density around the CTD and NTD of TRPV2APO_1 and TRPV2CBD_2.

Model representation of the distal CTDs of (A) left panel) TRPV2APO_1 (salmon) and (B) left panel) TRPV2CBD_2 (blue). This view is of the back of the region indicated by the black square on the overall models (A–B) center panels). Model representation of the distal NTD of (A) right panel) TRPV2APO_1 (salmon) and the distal CTD of (B) right panel) TRPV2CBD_2 (blue). This view is of the front of the region indicated by the black square on the overall models (A–B) center panels). Chain A of each model is colored salmon (TRPV2APO_1) or blue (TRPV2CBD_2). For both models, the adjacent chain B is colored wheat. Residues of interest are represented as sticks. Cryo-EM map density is contoured to σ = 5.

https://doi.org/10.7554/eLife.48792.022
Figure 4 with 2 supplements
Conformational changes between apo TRPV2 states.

(A) Overlay of the TRPV2APO_1 (salmon) and TRPV2APO_2 (purple) selectivity filter, viewed from the extracellular side of the membrane. (B) Zoom view of the overlay at S5-PH-S6. (C) Overlay of the TRPV2APO_1 (salmon) and TRPV2APO_2 (purple) structures, viewed from the extracellular side of the membrane. (D) Zoom view from the intracellular side of the membrane of the overlay of the TRPV2APO_1 (salmon) and TRPV2APO_2 (purple) structures. Residues of interest are represented as sticks and labeled. Dashed black lines are references to indicate rotation. The measurements for rotation and lateral shifts are labeled and indicated with black arrows.

https://doi.org/10.7554/eLife.48792.023
Figure 4—figure supplement 1
Cryo-EM map density around the selectivity filter of TRPV2APO_1 and TRPV2APO_2.

Density for (A) TRPV2APO_1 (salmon) and (B) TRPV2APO_2 (purple) is contoured to σ = 5. The selectivity filter is viewed from the extracellular side of the membrane. Residues of interest are represented as sticks.

https://doi.org/10.7554/eLife.48792.024
Figure 4—figure supplement 2
Conformational changes at the selectivity filter between TRPV1 apo and DkTx/RTX-bound structures in nanodiscs.

(A) Overlay of the TRPV1 apo state (PDB 5IRZ, gray) and TRPV1 DkTx/RTX-bound state (PDB 5IRX, orange) selectivity filter, viewed from the extracellular side of the membrane. (B) Zoom view of the overlay at S5-PH-S6. Residues of interest are represented as sticks and labeled. Shifts are with black arrows.

https://doi.org/10.7554/eLife.48792.025

Tables

Table 1
Cryo-EM data collection and model statistics.
https://doi.org/10.7554/eLife.48792.013
TRPV2APO_1
(EMD-20677,
PDB 6U84)
TRPV2APO_2
(EMD-20678,
PDB 6U85)
TRPV2CBD_1
(EMD-20686,
PDB 6U8A)
TRPV2CBD_2
(EMB-20682,
PDB 6U88)
Data collection and processing
Magnification81,000x81,000x
Detector modesuper-resolutioncounting
Voltage (kV)300300
Defocus range (μm)0.8–3.00.8–3.0
Pixel size (Å)1.061.06
Total extracted particles (no.)1,181,3472,290,820
Refined particles (no.)598,859710,728
Final particles (no.)42,40714,33223,94436,153
Symmetry imposedC4C4C4C4
Map sharpening B factor (Å2)−82−88−51−38
Map resolution (Å)
FSC threshold
3.7
0.143
4.0
0.143
3.4
0.143
3.2
0.143
Model Refinement
Model resolution cut-off (Å)3.74.03.43.2
Model composition
Nonhydrogen atoms
Protein residues
Ligands

20,460
2520
0

20,140
2472
0

20,036
2468
CBD: 4

20,016
2444
CBD: 4
R.M.S. deviations
Bond lengths (Å)
Bond angles (°)

0.011
0.742

0.007
0.664

0.008
0.947

0.009
1.060
Validation
MolProbity score
Clashscore
Poor rotamers (%)
CαBLAM outliers (%)
EMRinger score

1.69
5.36
0.90
1.14
1.72

2.06
11.75
0.18
0.84
3.03

1.37
3.39
0.37
0.50
2.61

1.38
4.23
0.55
2.18
2.55
Ramachandran plot
Favored (%)
Allowed (%)
Disallowed (%)

93.97
6.03
0.00

92.31
7.69
0.00

96.38
3.62
0.00

96.97
3.03
0.00

Data availability

cryoEM maps have been deposited in the Electron Microscopy Data Bank under the following accession codes: EMD-20677, EMD-20678, EMD-20686 and EMD-20682. The models built into the cryoEM maps have been deposited into the Protein Data Bank under the following accession codes: 6U84, 6U86, 6U8A and 6U88. The maps and models analyzed in this study are included with the manuscript and supporting files.

The following data sets were generated
  1. 1
    Electron Microscopy Data Bank
    1. RA Pumroy
    2. VY Moiseenkova-Bell
    (2019)
    ID EMD-20677. Apo full-length rat TRPV2 in nanodiscs, state 1.
  2. 2
    Protein Data Bank
    1. RA Pumroy
    2. VY Moiseenkova-Bell
    (2019)
    ID PDB 6U84. Apo full-length rat TRPV2 in nanodiscs, state 1.
  3. 3
    Electron Microscopy Data Bank
    1. RA Pumroy
    2. VY Moiseenkova-Bell
    (2019)
    ID EMD-20678. Apo full-length rat TRPV2 in nanodiscs, state 2.
  4. 4
    Protein Data Bank
    1. RA Pumroy
    2. VY Moiseenkova-Bell
    (2019)
    ID PDB 6U86. Apo full-length rat TRPV2 in nanodiscs, state 2.
  5. 5
    Electron Microscopy Data Bank
    1. RA Pumroy
    2. VY Moiseenkova-Bell
    (2019)
    ID EMD-20686. CBD-bound full-length rat TRPV2 in nanodiscs, state 1.
  6. 6
    Protein Data Bank
    1. RA Pumroy
    2. VY Moiseenkova-Bell
    (2019)
    ID PDB 6U8A. CBD-bound full-length rat TRPV2 in nanodiscs, state 1.
  7. 7
    Electron Microscopy Data Bank
    1. RA Pumroy
    2. VY Moiseenkova-Bell
    (2019)
    ID EMD-20682. CBD-bound full-length rat TRPV2 in nanodiscs, state 2.
  8. 8
    Protein Data Bank
    1. RA Pumroy
    2. VY Moiseenkova-Bell
    (2019)
    ID PDB 6U88. CBD-bound full-length rat TRPV2 in nanodiscs, state 2.

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