CryoEM structures of the human CLC-2 voltage-gated chloride channel reveal a ball-and-chain gating mechanism
- Department of Molecular and Cellular Physiology, Stanford University, United States
- Department of Chemistry, Stanford University, United States
- Department of Computer Science, Stanford University, United States
- Department of Structural Biology, Stanford University, United States
- Institute for Computational and Mathematical Engineering, Stanford University, United States
- Division of CryoEM and Bioimaging, SSRL, SLAC National Accelerator Laboratory, Stanford University, United States
- Department of Bioengineering and Department of Microbiology and Immunology, Stanford University, United States
Figures
Figure 1
Gating conformations in CLC transporters and channels.
(A) Cartoon depictions of the CLC anion permeation pathway. The ‘gating glutamate’ (Egate) can occupy anion-binding sites within the permeation pathway (‘down’ and ‘middle’ conformations, occupying …
Figure 2 with 4 supplements
CryoEM structure of the human CLC-2 channel.
Overall structure of the transmembrane domain (CLC2-TM) at 2.46 Å. The identical subunits of the homodimer are shown in purple and gray; Cl− ions are shown as green balls. (A) CryoEM density map …
Figure 2—figure supplement 1
Micrograph, 2D classes, and structure validation of CLC2-TM.
(A) Representative motion-corrected cryoEM micrograph. (B) 2D class averages. (C) Gold standard Fourier shell correlation (FSC) plots calculated in cryoSPARC. (D) Local resolution of the cryoEM map …
Figure 2—figure supplement 2
CryoEM workflow of the CLC2 single-particle cryoEM data processing.
A total of 14,198 movie stacks were collected on a 300 kV Titan Krios cryo-electron microscope. cryoSPRAC was used for 2D classification, and the CLC2-TM density map was obtained after 2D …
Figure 2—figure supplement 3
Helix map of CLC2-TM.
CryoEM densities and model of CLC-2 transmembrane helices (contour level: 0.73, 2.7 σ).
Figure 2—figure supplement 4
Egate position and Cl−-binding sites (Sext and Scen).
(A) Comparison between CLC-2 (purple), CLC-1 (light blue), and cmCLC (salmon pink). Egate (E205 in CLC-2, E232 in CLC-1, and E210 in cmCLC), SerC (S162 in CLC2, S189 in CLC-1, and S165 in cmCLC), …
Figure 3 with 2 supplements
Pore profile detected in CLC-2.
(A) Left: The primary (orange) and secondary (yellow) pore detected in CLC-2 using Caver. Middle: The detected pore radii are displayed in dots as a color map. Right: Zoomed-in view of the …
Figure 3—figure supplement 1
Cl− pathway in CLC-2 and CLC-1.
(A) CLC-2 Cl− pathway as shown in Figure 3 but omitting sections with pore radius less than 1 Å. Egate, occupying the Scen site, blocks the canonical Cl− pathway. (B) Same as panel A, for CLC-1 (PDB …
Figure 3—figure supplement 2
Comparison of primary and secondary Cl− pathways with transporter Cl− and H+ pathways.
(A) The top two panels are repeated from Figure 3A, indicating the primary (orange) and secondary (yellow) caver-detected pores in CLC-1 and CLC-2. The lower panel shows CLC-ec1 (PDB ID: 1OTS), a …
Figure 4 with 5 supplements
Different conformations of the C-terminal domain (CTD).
(A) Overall structure of the two CLC-2 conformations. Left: CLC2-CTDsym at 2.75 Å. Right: CLC2-CTDasym at 2.76 Å. The subunits of the homodimer are shown in orange (CLC2-CTDsym) or yellow …
Figure 4—figure supplement 1
Structure validation of CLC2-CTDsym and CLC2-CTDasym.
(A) Gold standard Fourier shell correlation (FSC) plots calculated in cryoSPARC for CLC2-CTDsym. (B) Local resolution of the cryoEM map of the CLC2-CTDsym. (C) Model validation using Q-scores of …
Figure 4—figure supplement 2
CryoEM density maps for the two C-terminal domain (CTD)-containing CLC-2 conformations, overlaid with structural models.
(A) CLC2-CTDsym (contour level: 0.68, 4.9 σ). (B) CLC2-CTDasym (contour level: 0.75, 6.8 σ). Side view (left) and top view (right) are shown. (C) CTD of CLC2-CTDsym shows two CBS domains on each …
Figure 4—figure supplement 3
Molecular dynamics (MD) analysis indicates conformational flexibility of the C-terminal domain (CTD).
(A) The CTD is highly mobile relative to the transmembrane domain, and often tilts upward or downward relative to its initial location. In the inset images, two representative frames from simulation …
Figure 4—figure supplement 4
Comparison of C-terminal domain (CTD) orientation with respect to the transmembrane (TM) domain.
(A) The CLC-1 structure is shown in light blue. The angle of orientation for the CTDs was calculated using the far end residue of helix T (the second helix of first CBS domain) as the endpoint of …
Figure 4—figure supplement 5
ATP-binding site comparison between CLC-2 and CLC-5.
Top: Structural overlay of CLC2-CTDsym (in orange) and CLC5-CTD (in dark purple) with ATP (pink carbon atoms; CPK coloring on non-carbon atoms; PDB ID: 2J9L). Bottom: Zoomed-in stereo view of …
Figure 5 with 6 supplements
The blocking hairpin structure corresponds to an N-terminal sequence of CLC-2.
(A) Left: The hairpin structure seen in the cryoEM density (blue mesh) fits N-terminal residues 14–28 (blue). Right: The subunit in CLC2-CTDasym structure that contains a C-terminal domain (CTD) …
Figure 5—figure supplement 1
Comparison of peptide candidates fitting into the hairpin density.
(A) Best-fitting peptide candidate from the N-terminus (left shown in blue) and from the C-terminus (right shown in purple) fitted into cryoEM hairpin density (blue mesh). (B) Q-score plot of the …
Figure 5—figure supplement 2
N-terminal hairpin sequence is conserved in CLC-2, but not other CLCs.
(A) Alignment of the N-terminal hairpin sequence (blue boxed) among different species. (B) Alignment of N-terminal hairpin sequence (blue boxed) among other human CLCs.
Figure 5—figure supplement 3
Electrostatic map of the N-terminal hairpin-blocking site.
Left: View from the cytoplasm showing the electrostatic surface potential of the transmembrane (TM) region (blue, positive; red, negative), with the hairpin shown in cartoon. Right: Side view …
Figure 5—figure supplement 4
Molecular dynamics (MD) simulations confirm the N-terminal hairpin structure is stable.
(A) In simulations, the N-terminus (blue) remains stably bound to the intracellular side of the transmembrane domain (orange). In the inset image, frames of the N-terminus taken every 200 ns from a …
Figure 5—figure supplement 5
The N-terminal hairpin blocks the primary Cl− pore in CLC-2.
(A) Left: Side view of CLC2-TM showing the caver-detected pore (primary in orange and secondary in yellow). Right: View of the primary pore from the intracellular side. (B) The N-terminal hairpin …
Figure 5—figure supplement 6
Mapping mutant data onto the cryoEM structure.
(A) Left: Same as Figure 5C, showing hairpin interactions. Right: Rotated view highlighting ball ‘receptor’ residues identified by Jordt and Jentsch, 1997. (B) Residues mutated in human …
Figure 6 with 2 supplements
Patch-clamp experiments support CLC-2 channel block by the N-terminal hairpin structure.
(A) Representative currents from WT and Delta-N CLC-2, recorded using the whole-cell patch-clamp configuration in response to the voltage protocol shown. (B) WT CLC-2 recording from panel A, shown …
-
Figure 6—source data 1
Original file for the Western blot analysis in Figure 6D (anti-GFP).
- https://cdn.elifesciences.org/articles/90648/elife-90648-fig6-data1-v1.zip
-
Figure 6—source data 2
PDF containing Figure 6D and original scan of the Western blot (anti-GFP) with highlighted band and sample labels.
- https://cdn.elifesciences.org/articles/90648/elife-90648-fig6-data2-v1.zip
-
Figure 6—source data 3
Excel file containing numerical data for electrophysiology and Western blot results shown in Figure 6C–F.
- https://cdn.elifesciences.org/articles/90648/elife-90648-fig6-data3-v1.zip
Figure 6—figure supplement 1
Data traces from the CLC-2 run-up experiments summarized in Figure 6G.
For Delta-N, separate scale bars indicate the currents for the top six and bottom 10 traces. AK-42 leak currents, not subtracted in these raw data traces, are summarized in Figure 6—source data 3.
Figure 6—figure supplement 2
CLC-2 current rectification.
(A) Example traces from two experiments on WT CLC-2. Current values at end of the −100-mV test pulses and in the steady-state portion of the +80-mV tail pulse (following the +20-mV test pulse) are …
Figure 7 with 9 supplements
Bound AK-42 blocks the Cl− pathway in CLC-2.
(A) Transmembrane (TM) domain structure of the CLC-2 structure in complex with AK-42 (CLC2-TM-AK42) at 2.74 Å. The identical subunits of the homodimer are shown in hot pink and gray; Cl− ions are …
Figure 7—figure supplement 1
CryoEM workflow of the CLC2-TM-AK42 single-particle cryoEM data processing.
A total of 14,300 movie stacks were collected on a 300 kV Titan Krios cryo-electron microscope. cryoSPRAC was used for 2D classification and CLC2-TM-AK42 density map was obtained after 2D …
Figure 7—figure supplement 2
Micrograph and 2D classes and structure validation of CLC2-AK42.
(A) Representative motion-corrected cryoEM micrograph. (B) 2D class averages. (C) Gold standard Fourier shell correlation (FSC) plots calculated in cryoSPARC for CLC2-TM-AK42 (left) and …
Figure 7—figure supplement 3
Model validation using Q-scores.
(A) Q-scores of CLC2-TM-AK42 for subunits A (left) and B (right). (B) Q-scores of CLC2-AK42-CTD-only for subunits A (left) and B (right). The black line represents the expected Q-score at respective …
Figure 7—figure supplement 4
cryoEM map and structure of CLC2-AK42.
(A) CryoEM model of CLC2-TM-AK42 with docked C-terminal domain (CTD). The transmembrane (TM) region is shown in hot pink and gray. The CTD is shown in green and gray. AK-42 is shown in yellow and …
Figure 7—figure supplement 5
Helix map of CLC2-TM-AK42.
CryoEM densities and model of CLC2-AK42 transmembrane helices (contour level: 0.52, 3.4 σ).
Figure 7—figure supplement 6
Density overlay at key Cl− pathway residues.
Zoomed-in view highlighting residues S162, E205, Y553, and the bound chloride ion, with cryoEM density overlay (contour level: 1.4, 9.0 σ).
Figure 7—figure supplement 7
AK-42-binding site comparison between CLC2-TM and CLC2-TM-AK42.
Top: Stereo view of structural overlay at the AK-42-binding site. Hydrogen bonds between AK-42 and residues K204, S392, K394 are indicated by blue dashed lines. Bottom: A view highlighting the …
Figure 7—figure supplement 8
Structural comparison of CLC2-TM and CLC2-TM-AK42.
Difference distance matrices comparing Cɑ residues on transmembrane (TM) helices between CLC2-TM and CLC2-TM-AK42 in subunits A (A) or B (B). Residues in loops connecting helices, where there is low …
Figure 7—figure supplement 9
AK-42-binding site comparison between CLC-2 and CLC-1.
(A) Top: Stereo view of the AK-42-binding site in CLC2-TM-AK42 (hot pink) overlaid with the same region in CLC-1 (blue, PDB:6coy). Residue numbers correspond to those of CLC-2. The five residues …
Figure 8
CLC-2’s gating is illustrated based on our new structural data.
Opening of each protopore requires at least two events – unblock by the N-terminal hairpin (N-ter) and rotation of Egate away from the permeation pathway. The dashed green arrow represents the Cl− …
Videos
Video 1
CLC-2 pore detected using Caver.
Video 2
CLC-1 pore detected using Caver.
Video 3
CLC-ec1 caver-detected pathway.
Video 4
Rigid body movement from CLC2-CTDsym to CLC2-CTDasym.
Video 5
Hairpin surface potential.
Video 6
AK-42 density.
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/90648/elife-90648-mdarchecklist1-v1.pdf
-
Supplementary file 1
cryoEM statistics.
- https://cdn.elifesciences.org/articles/90648/elife-90648-supp1-v1.docx
