Cryo-EM structures of an LRRC8 chimera with native functional properties reveal heptameric assembly
- Department of Molecular Physiology and Biophysics, School of Medicine, Vanderbilt University, United States
- Center for Structural Biology, Vanderbilt University, United States
- Department of Anesthesiology, Vanderbilt University Medical Center, United States
- Department of Pharmacology, School of Medicine, Vanderbilt University, United States
Figures
Figure 1 with 1 supplement
Construct design of 8C-8A(IL125).
(A) Sequence alignment of LRRC8A and LRRC8C around the swapped IL125 region. The swapped region in the 8C-8A(IL125) construct is shown inside a black box. The key below amino acid sequences denotes …
Figure 1—figure supplement 1
Purification of 8C-8A(IL125).
SDS-PAGE (A), native-PAGE (B), and size exclusion chromatography (C) analysis of the purified LRRC8A and 8C-8A(IL125) proteins.
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Figure 1—figure supplement 1—source data 1
Raw and annotated images of the SDS-PAGE gel analyzing the purified LRRC8A and 8C-8A(IL125) proteins.
- https://cdn.elifesciences.org/articles/82431/elife-82431-fig1-figsupp1-data1-v2.zip
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Figure 1—figure supplement 1—source data 2
Raw and annotated images of the native-PAGE gel analyzing the purified LRRC8A and 8C-8A(IL125) proteins.
- https://cdn.elifesciences.org/articles/82431/elife-82431-fig1-figsupp1-data2-v2.zip
Figure 2 with 3 supplements
Cryo-electron microscopy (cryo-EM) structure of 8C-8A(IL125).
(A–B) Cryo-EM maps of 8C-8A(IL125) class 1 structure viewed through the membrane plane (A) and from the cytoplasm (B). (C) Ribbon representation of the 8C-8A(IL125) class 1 structure viewed through …
Figure 2—figure supplement 1
Cryo-electron microscopy (cryo-EM) analysis of 8C-8A(IL125).
Flowchart detailing the particle selection and refinement procedure to obtain the cryo-EM maps of 8C-8A(IL125). Local resolution maps are shown on maps viewed at two different thresholds in Chimera. …
Figure 2—figure supplement 2
Cryo-electron microscopy (cryo-EM) analysis of 8C-8A(IL125).
(A) Cryo-EM map of 8C-8A(IL125) class 1 structure colored by individual subunits. (B) Masks used for local refinement of individual subunits are shown as transparent gray surfaces. Each mask was …
Figure 2—figure supplement 3
Structural comparison of 8C-8A(IL125) to LRRC8A and pannexins.
(A–C) Ribbon representations of LRRC8A (PBD ID: 5ZSU) (A), 8C-8A(IL125) class 1 structure (B), and pannexin 1 (PDB ID: 6VD7) (C) viewed from the extracellular space (top), through the membrane plane …
Figure 3 with 3 supplements
Subunit arrangement of the 8C-8A(IL125) chimera.
(A–B) Surface representation of the 8C-8A(IL125) class 1 structure viewed from two sides, highlighting the ‘tight’ (A) and ‘loose’ (B) interfaces. (C–E) Intracellular domain (ICD), transmembrane …
Figure 3—figure supplement 1
Comparison of subunit interfaces.
(A) Overlay of two 8C-8A(IL125) subunit pairs of the class 1 structure that form tight and loose interfaces. Structures were aligned on their extracellular domains (ECDs). (B–C) Close-up view of the …
Figure 3—figure supplement 2
Comparison of subunit interfaces.
(A) Overlay of two 8C-8A(IL125) subunit pairs of the class 1 structure that form tight and loose interfaces. Structures were aligned on their extracellular domains (ECDs). (B–C) Close-up view of the …
Figure 3—figure supplement 3
Comparison of subunit interfaces.
(A) Overlay of two 8C-8A(IL125) subunit pairs of the class 1 structure that form tight and loose interfaces. Structures were aligned on their extracellular domains (ECDs). (B–C) Close-up view of the …
Figure 4
Structural heterogeneity of 8C-8A(IL125) protomers.
(A) Structural comparison of the 8C-8A(IL125) subunits in the class 1 structure. The structures are aligned based on their extracellular domains (ECDs). (B) Close-up view of the box region in panel …
Figure 5 with 2 supplements
Conformational heterogeneity of 8C-8A(IL125) chimeras.
(A–B) Cryo-electron microscopy (cryo-EM) maps (before sharpening) of 8C-8A(IL125) 3D classes viewed through the membrane plane (A) and from the cytoplasm (B). Individual subunits are colored as in Fi…
Figure 5—figure supplement 1
Comparison of 8C-8A(IL125) structures.
(A) Overlay of 8C-8A(IL125) structures in different classes viewed through the membrane plane. (B–D) Overlay of 8C-8A(IL125) structures in different classes viewed from the intracellular space …
Figure 5—figure supplement 2
Comparison of the D40-K51 loop among different classes.
Close-up view of the pore around the residues D50 and K51, which are shown as sticks. The residues that adopt different conformations compared to others are labeled in red. The dashed circle …
Figure 6 with 2 supplements
Comparison of channel pores.
(A–B) Pore openings of the 8C-8A(IL125) heptameric channel (class 1 structure) (A) and LRRC8A (PDB ID: 5ZSU) homohexameric channel (B) calculated using the software program HOLE (Smart et al., 1996).…
Figure 6—figure supplement 1
Effect of intracellular ionic strength on activation of LRRC8A and the 8C-8A(IL125) chimeric channel.
The relative current was quantified as the fold change in current measured immediately after whole-cell access was achieved and currents measured 120 s later. As we have shown previously (Yamada et …
Figure 6—figure supplement 2
Heptameric oligomerization leads to larger pore sizes.
(A–C) Close-up cytoplasmic view of the narrowest constriction sites of 8C-8A(IL125) class 1 structure (A), LRRC8A (PDB ID: 5ZSU) (B), and LRRC8D (PDB ID: 6M04) (C). (D) The heptameric LRRC8A and …
Figure 7 with 1 supplement
Interaction of lipids with the 8C-8A(IL125) chimera.
(A) Ribbon representation of the 8C-8A(IL125) class 1 structure along with the lipid-like cryo-electron microscopy (cryo-EM) densities (yellow) between the subunits viewed through the membrane …
Figure 7—figure supplement 1
Cryo-electron microscopy (cryo-EM) analysis of 8C-8A(IL125) reconstituted in nanodiscs.
(A) Flowchart detailing the particle selection and refinement procedure to obtain the cryo-EM maps of the 8C-8A(IL125) nanodiscs. FSC curves and angular distributions are shown. (B–C) Full (B) and …
Tables
Table 1
Cryo-electron microscopy (cryo-EM) data collection, refinement, and validation statistics.
| Data collection and processing | |||||
|---|---|---|---|---|---|
| Microscope | FEI Krios G3i microscope | ||||
| Detector | Gatan K3 direct electron camera | ||||
| Nominal magnification | ×81,000 | ||||
| Voltage (kV) | 300 | ||||
| Electron exposure (e/Å2) | 54 | ||||
| Defocus range (µm) | –0.8 to –1.5 | ||||
| Pixel size (Å) | 1.1 | ||||
| Number of Micrographs | 3198 | ||||
| Particles images (no.) | 846,122 | ||||
| Conformational state | Class 1 | Class 2 | Class 3 | Class 4 | Class 5 |
| Symmetry imposed | C1 | C1 | C1 | C1 | C1 |
| Final particles images (no.) | 203,011 | 132,722 | 100,772 | 93,179 | 85,591 |
| Map resolution (Å) (FSC threshold = 0.143) | 3.4 | 3.6 | 3.7 | 3.8 | 4.0 |
| Refinement | |||||
| Model resolution (Å) (original map, FSC threshold = 0.5) | 3.6 | 3.9 | 3.9 | 4.2 | 4.4 |
| B-factor used for map sharpening (Å2) | –102.0 | –88.2 | –84.7 | –70.5 | –83 |
| Model composition | |||||
| Non-hydrogen atoms | 17,499 | 17,499 | 17,465 | 10,435 | 10,435 |
| Protein residues | 2101 | 2101 | 2097 | 2101 | 2101 |
| Mean B factors (Å2) | |||||
| Protein | 32.4 | 23.5 | 32.4 | 44.9 | 38.34 |
| R.m.s. deviations | |||||
| Bond lengths (Å) | 0.003 | 0.002 | 0.003 | 0.002 | 0.004 |
| Bond angles (°) | 0.563 | 0.525 | 0.553 | 0.516 | 0.807 |
| Molprobity score | 1.72 | 1.68 | 1.75 | 1.37 | 1.49 |
| Clash score | 5.04 | 4.84 | 6.02 | 1.14 | 1.54 |
| Poor rotamers (%) | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
| Favored (%) | 92.9 | 93.5 | 93.6 | 89.95 | 88.3 |
| Allowed (%) | 7.1 | 6.5 | 6.4 | 10.0 | 11.6 |
| Disallowed (%) | 0 | 0 | 0 | 0.05 | 0.1 |
Table 2
Total buried solvent-accessible surface area between subunits for each domain.
| Buried surface area between the neighboring subunits (Å2)* | |||||||
|---|---|---|---|---|---|---|---|
| Subunits A-B | Subunits B-C | Subunits C-D | Subunits D-E | Subunits E-F | Subunits F-G | Subunits G-A | |
| Tight | Tight | Tight | Loose | Tight | Tight | Loose | |
| ECD† | 1995 | 1897 | 1964 | 1905 | 1900 | 1896 | 1869 |
| TMD† | 664 | 625 | 620 | 122 | 660 | 670 | 99 |
| ICD† | 462 | 294 | 500 | 0 | 559 | 502 | 0 |
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*
Buried solvent-accessible surface area calculations were performed using the software NACCESS v2.1.1 (Hubbard and Thornton, 1993).
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†
Domain definitions used for these calculations are as follows: ECD: residues 49–121 and 288–310; TMD: residues 20–48, 122–150, 260–287, and 311–342; ICD: residues 151–259 and 343–405.
Table 3
Composition of patch pipette and solutions.
| Patch pipette solutions | Bath solutions | ||||
|---|---|---|---|---|---|
| Control | Control | Control | Hypotonic | ||
| CsCl | 126 mM | 26 mM | 75 mM | 75 mM | |
| Cesium methanesulfonate | 100 mM | ||||
| MgSO4 | 2 mM | 2 mM | 5 mM | 5 mM | |
| Ca-gluconate2 | 1 mM | 1 mM | |||
| ATP-Na2 | 2 mM | 2 mM | |||
| GTP-Na2 | 0.5 mM | 0.5 mM | |||
| Glutamine | 2 mM | 2 mM | |||
| EGTA | 1 mM | 1 mM | |||
| HEPES | 20 mM | 20 mM | 12 mM | 12 mM | |
| Tris | 8 mM | 8 mM | |||
| CsOH | 12 mM | 12 mM | |||
| HCl | 2 mM | 2 mM | |||
| Glucose | 5 mM | 5 mM | |||
| Sucrose | 16 mM | 16 mM | 115 mM | 70 mM | |
| pH* | 7.2 | 7.2 | 7.4 | 7.4 | |
| Osmolality | 275 mOsm | 275 mOsm | 300 mOsm | 250 mOsm | |
| Ionic strength | 0.162 M | 0.162 M | |||
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*
The pH of patch pipette and bath solutions was adjusted with CsOH and HCl, respectively.
