Structural features of heteromeric channels composed of CALHM2 and CALHM4 paralogs
- Department of Biochemistry, University of Zurich, Switzerland
Figures
Figure 1 with 1 supplement
Heteromerization and functional properties of placental calcium homeostasis modulators (CALHM) paralogs.
(A) Western blot (detecting the myc-tag attached to the C-termini of respective constructs) of CALHM subunits expressed upon co-transfection of HEK293S GnTI− cells with pairs of CALHM subunits. …
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Figure 1—source data 1
Uncropped images of immunoblots and raw data plotted in Figure 1A.
- https://cdn.elifesciences.org/articles/96138/elife-96138-fig1-data1-v1.xlsx
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Figure 1—source data 2
Raw recordings displayed and plotted in Figure 1B–F.
- https://cdn.elifesciences.org/articles/96138/elife-96138-fig1-data2-v1.xlsx
Figure 1—figure supplement 1
Electrophysiology data of calcium homeostasis modulators (CALHM) paralogs recorded at low extracellular Ca2+.
(A–D) Patch clamp electrophysiology recordings (whole-cell configuration) of representative traces of indicated CALHM subunits expressed in HEK-293 cells measured in buffers not containing Ca2+ on …
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Figure 1—figure supplement 1—source data 1
Raw recordings displayed and plotted in Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/96138/elife-96138-fig1-figsupp1-data1-v1.xlsx
Figure 2 with 2 supplements
Structural characterization of the CALHM4/SbC4 complex.
(A) Cryo-electron microscopy (Cryo-EM) density at 3.7 Å (after application of D10 symmetry) of a pair of decameric CALHM4/SbC4 complexes interacting via their cytoplasmic regions. (B) Ribbon …
Figure 2—figure supplement 1
Sybody selection and characterization.
(A) Sequence alignment of the two sybodies SbC4 and SbC2 specifically targeting CALHM4 and CALHM2, respectively. (B) Size exclusion chromatography profile of the CALHM4/SbC4 complex. The elution …
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Figure 2—figure supplement 1—source data 1
SDS-PAGE gel images and size-exclusion chromatography data plotted in Figure 2—figure supplement 1B, E.
- https://cdn.elifesciences.org/articles/96138/elife-96138-fig2-figsupp1-data1-v1.xlsx
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Figure 2—figure supplement 1—source data 2
Surface plasmon resonance (SPR) binding data plotted in Figure 2—figure supplement 1C, D, F, G.
- https://cdn.elifesciences.org/articles/96138/elife-96138-fig2-figsupp1-data2-v1.xlsx
Figure 2—figure supplement 2
Cryo-electron microscopy (Cryo-EM) reconstruction of CALHM4 in complex with sybody SbC4.
(A) Representative cryo-EM micrograph acquired with a Titan Krios microscope equipped with a K3 camera. (B) Representative 2D class averages of the CALHM4/SbC4 complex. (C) Data processing workflow. …
Figure 3 with 1 supplement
Structural characterization of a CALHM2/SbC2 complex.
(A) Cryo-electron microscopy (Cryo-EM) density of the undecameric CALHM2/SbC2 complex at 3.1 Å (after application of C11 symmetry). (B) Ribbon representation of the same complex viewed parallel to …
Figure 3—figure supplement 1
Cryo-electron microscopy (Cryo-EM) reconstruction of CALHM2 in complex with sybody SbC2.
(A) Representative cryo-EM micrograph acquired with a Titan Krios microscope equipped with a K3 camera. (B) 2D class averages of CALHM2/SbC2 complex. (C) Data processing workflow. Particles were …
Figure 4 with 2 supplements
Classification of CALHM2/CALHM4 heteromers.
Classes correspond to distinct populations of oligomers in the CALHM2/4/SbC2/SbC4 complex. (A) Assemblies containing an excess of CALHM4 subunits. Shown is the well-resolved halve of interacting …
Figure 4—figure supplement 1
Cryo-electron microscopy (Cryo-EM) reconstruction of CALHM2/4 in complex with sybody SbC4.
(A) Representative cryo-EM micrograph acquired with a Titan Krios microscope equipped with a K3 camera. (B) Data processing workflow. Particles were picked and subjected to 2D classification. 2D …
Figure 4—figure supplement 2
Cryo-electron microscopy (Cryo-EM) reconstruction of CALHM2/4 in complex with sybodies SbC2 and SbC4.
(A) Representative cryo-EM micrograph acquired with a Titan Krios microscope equipped with a K3 camera. (B) Data processing workflow. Particles were picked and subjected to 2D classification. …
Figure 5
CALHM2/CALHM4 heteromer structure.
(A) Schematic depiction of the subunit distribution in CALHM2/4 heteromers. 4 refers to CALHM4, 2 to CALHM2 in ‘up’ conformation, 2d to CALHM2 in ‘down’ conformation, 2 u/d to a mixed population of …
Figure 6
Conformational properties of calcium homeostasis modulators (CALHM) subunits in CALHM2/4 heteromers.
(A) Cryo-electron microscopy (Cryo-EM) density of CALHM2 subunits in ‘down’ (position 3, top) and ‘up’ (position 6, left) and of CALHM4 (in ‘down’ conformation, position 1, bottom). (B) Ribbon …
Figure 7
Structural features of subunit interfaces.
(A–C) Interaction interfaces between pairs of CALHM2 subunits (A), CALHM4 subunits (B), and a CALHM2 and a CALHM4 subunit (C). Shown are ribbon representations of neighboring subunits viewed from …
Figure 8
Discussion.
(A, B) Superposition of single subunits of different calcium homeostasis modulators (CALHM) paralogs on CALHM2 viewed from within the membrane (top) and of their cytosolic domain viewed from the …
Author response image 1
Classification of subunit pairs of undecameric CALHM2/4 channels bound to sybodies SbC2 and SbC4 after the processing combining symmetry expansion and partial signal subtraction.
(A) Classes showing CALHM2 subunit pairs. (B) Classes showing subunits at interfaces to CALHM4.
Tables
Table 1
Cryo-electron microscopy (Cryo-EM) Data collection, refinement, and validation statistics.
| CALHM4/SbC4 | CALHM2/SbC2 | CALHM2/CALHM4/SbC4 | CALHM2/CALHM4/SbC2/SbC4 | |
|---|---|---|---|---|
| EMD-19365PDB 8RMN | EMD-19362PDB 8RMK | EMD-19363PDB 8RML | EMD-19364PDB 8RMM | |
| Data collection and processing | ||||
| Microscope | FEI Titan Krios G3i | FEI Titan Krios G3i | FEI Titan Krios G3i | FEI Titan Krios G3i |
| Camera | Gatan K3 +GIF | Gatan K3 +GIF | Gatan K3 +GIF | Gatan K3 +GIF |
| Magnification | 130,000 | 130,000 | 130,000 | 130,000 |
| Voltage (kV) | 300 | 300 | 300 | 300 |
| Electron exposure (e–/Å2) | 70 | 72 | 60 | 60 |
| Defocus range (μm) | –1.0 to –2.4 | –1.0 to –2.4 | –1.0 to –2.4 | –1.0 to –2.4 |
| Pixel size* (Å) | 0.651 (0.3255) | 0.651 (0.3255) | 0.651 (0.3255) | 0.651 (0.3255) |
| Initial particle images (no.) | 424,942 | 1,068,223 | 1,345,836 | 2,800,265 |
| Final particle images (no.) | 52,248 | 37,894 | 131,652 | 93,191 |
| Symmetry imposed | D10 | C11 | C1 | C1 |
| Map resolution (Å) | ||||
| FSC threshold 0.143 | 3.7 | 3.07 | 3.84 | 3.26 |
| Map resolution range (Å) | 3.5–5 | 2.6–5 | 3.5–6 | 3–6 |
| Refinement | ||||
| Model resolution (Å) | ||||
| FSC threshold 0.5 | 3.9 | 3.2 | 4.1 | 3.7 |
| Map sharpening b-factor (Å2) | –152.97 | –101.8 | –113.8 | –67.2 |
| Model composition | ||||
| Non-hydrogen atoms | 55,460 | 36,113 | 24,456 | 32,891 |
| Protein residues | 7199 | 4521 | 3089 | 4163 |
| Ligand (PLC) | 11 | 0 | 6 | |
| B factors (Å2) | ||||
| Protein | 74.47 | 75.52 | 121.74 | 69.29 |
| Ligand | 59.93 | 80.08 | ||
| R.M.S. deviations | ||||
| Bond lengths (Å) | 0.004 | 0.003 | 0.004 | 0.003 |
| Bond angles (°) | 0.710 | 0.611 | 0.596 | 0.671 |
| Validation | ||||
| MolProbity score | 2.00 | 1.86 | 1.87 | 1.85 |
| Clashscore | 13.63 | 12.04 | 12.92 | 11.37 |
| Poor rotamers (%) | 1.16 | 0.45 | 0.46 | 0.79 |
| Ramachandran plot | ||||
| Favored (%) | 95.60 | 96.05 | 96.28 | 96 |
| Allowed (%) | 4.40 | 3.95 | 3.72 | 3.98 |
| Disallowed (%) | 0.00 | 0.00 | 0.00 | 0.02 |
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*
Values in parentheses indicate the pixel size in super-resolution.
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | MC1016 | Sigma | C66303 | |
| Cell line (Homo-sapiens) | HEK293S GnTI- | ATCC | CRL-3022 | |
| Cell line (Homo-sapiens) | HEK-293 | ATCC | CRL-1573 | |
| Antibody | Anti-c-Myc (Mouse monoclonal) | Sigma | M4439 | (1:5000) |
| Antibody | Peroxidase AffiniPure Goat Anti-Mouse IgG H+L (Goat polyclonal) | Jackson ImmunoResearch | 115-035-146 | (1:10000) |
| Recombinant DNA reagent | Modified mammalian pcDNA 3.1 (+) expression vector for FX cloning system, C-terminal 3 C cleavage site, myc tag and 6x HisTag | Dutzler group | N/A | |
| Recombinant DNA reagent | Modified mammalian pcDNA 3.1 (+)expression vector for FX cloning system, C-terminal 3 C cleavage site, Venus fluorescent tag, myc tag and SBP tag | Dutzler group | N/A | |
| Recombinant DNA reagent | pBXNPHM3 (plasmid) | Seeger group | Addgene #110099 | |
| Recombinant DNA reagent | pSbinit (plasmid) | Seeger group | Addgene #110100 | |
| Recombinant DNA reagent | Homo sapiens CALHM1 | GenScript | Accession NM_001001412.3 | |
| Recombinant DNA reagent | Homo sapiens CALHM2 | GenScript | Accession NM_015916.5 | |
| Recombinant DNA reagent | Homo sapiens CALHM3 | GenScript | Accession NM_001129742.2 | |
| Recombinant DNA reagent | Homo sapiens CALHM4 | GenScript | Accession NM_001366078.1 | |
| Recombinant DNA reagent | Homo sapiens CALHM6 | GenScript | Accession NM_001010919.3 | |
| Commercial assay or kit | Amersham ECL Prime Western Blotting Detection Kit | GE Healthcare | RPN2232 | |
| Commercial assay or kit | EZ-link NHS-PEG4-biotin | Sigma | A39259 | |
| Chemical compound, drug | Benzamidine | Sigma | B6506 | |
| Chemical compound, drug | Calcium chloride | Sigma | 223506 | |
| Chemical compound, drug | Chloramphenicol | Sigma | C1919 | |
| Chemical compound, drug | D-desthiobiotin | Sigma | D1411 | |
| Chemical compound, drug | DNase I | Sigma | EN0521 | |
| Chemical compound, drug | Dulbecco’s Modified Eagle’s Medium (DMEM) High glucose, pyruvate | Sigma | D6429 | |
| Chemical compound, drug | EGTA | Sigma | 03777 | |
| Chemical compound, drug | Fetal bovine serum | Sigma | F7524 | |
| Chemical compound, drug | Glucose | AppliChem | A1422.1000 | |
| Chemical compound, drug | Glycerol 99% | Sigma | G7757 | |
| Chemical compound, drug | Glycol-diosgenin (GDN) | Anatrace | GDN101 | |
| Chemical compound, drug | HCl | Millipore | 1.00319.1000 | |
| Chemical compound, drug | HEPES | Sigma | H3375 | |
| Chemical compound, drug | HyClone HyCell TransFx-H medium | Cytiva | SH30939.02 | |
| Chemical compound, drug | Imidazole | Roth | X998.4 | |
| Chemical compound, drug | Kolliphor P188 | Sigma | K4894 | |
| Chemical compound, drug | L-(+)-arabinose | Sigma | A3256 | |
| Chemical compound, drug | L-glutamine | Sigma | G7513 | |
| Chemical compound, drug | Lauryl Maltose Neopentyl Glycol (LMNG) | Anatrace | NG310 | |
| Chemical compound, drug | Leupeptin | AppliChem | A2183.0100 | |
| Chemical compound, drug | Lysozyme | AppliChem | A3711.0050 | |
| Chemical compound, drug | Magnesium chloride | Fluka | 63.065 | |
| Chemical compound, drug | Penicillin-streptomycin | Sigma | P0781 | |
| Chemical compound, drug | Pepstatin A | Axon lab | A2205.0100 | |
| Chemical compound, drug | Phenylmethylsulfonyl fluoride (PMSF) | Sigma | PMSF-RO | |
| Chemical compound, drug | Polyethylenimine HCl MAX, Linear, MW 40,000 (PEI MAX 40000) | Chemie Brunschwig AG | POL24765 | |
| Chemical compound, drug | Potassium chloride | Sigma | 746346 | |
| Chemical compound, drug | RNase | Sigma | R5125 | |
| Chemical compound, drug | Sodium chloride | Sigma | 71380 | |
| Chemical compound, drug | Sodium hydroxide | Sigma | S8045 | |
| Chemical compound, drug | Terrific broth | Sigma | T9179 | |
| Chemical compound, drug | Tris | AppliChem | A1379 | |
| Chemical compound, drug | Tween 20 | Sigma | 93773 | |
| Chemical compound, drug | Valproic acid sodium salt | Sigma | P4543 | |
| Software, algorithm | Axon Clampex 10.6 | Molecular Devices | N/A | |
| Software, algorithm | Axon Clampfit 11.0.3 | Molecular Devices | N/A | |
| Software, algorithm | Chimera 1.16 | Pettersen et al., 2004 | http://www.cgl.ucsf.edu/chimera/ | |
| Software, algorithm | ChimeraX 1.3 | Pettersen et al., 2021 | https://www.cgl.ucsf.edu/chimerax/ | |
| Software, algorithm | Coot 0.9.8.91 | Emsley and Cowtan, 2004 | https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ | |
| Software, algorithm | cryoSPARC v3.2.0–4.0 | Structural Biotechnology Inc. | RRID:SCR_016501 | |
| Software, algorithm | CTFFIND4.1 | Rohou and Grigorieff, 2015 | http://grigoriefflab.janelia.org/ctf | |
| Software, algorithm | DINO 0.9.4 | http://www.dino3d.org | http://www.dino3d.org | |
| Software, algorithm | EPU 2.9 | Thermo Fisher Scientific | N/A | |
| Software, algorithm | PHENIX 1.14 | Afonine et al., 2018 | http://phenix-online.org/ | |
| Software, algorithm | RELION-4.0 | Scheres, 2012 | https://www2.mrc-lmb.cam.ac.uk/relion/ | |
| Software, algorithm | Prism 10 | GraphPad | https://www.graphpad.com/ | |
| Other | 200 mesh Au 1.2/1.3 cryo-EM grids | Quantifoil | N1-C14nAu20-01 | See Methods, Cryo-EM sample preparation, and data collection |
| Other | 0.22 µm Ultrafree-MCCentrifugal Filter | Millipore | UFC30GV | See Methods, CALHM protein purification |
| Other | Amicon 100 kDa MWCO centrifugal filter | Millipore | UFC810096 | See Methods, CALHM protein purification |
| Other | NiNTA agarose beads | ABT | 6BCL-NTANi-100 | See Methods, CALHM protein purification, Sybody expression, and purification |
| Other | PD-10 desalting column | Sigma | GE17-0851-01 | See Methods, Sybody selection |
| Other | SRT-10C SEC 100 | Sepax Technologies | 239100–10,030 | See Methods, Sybody expression and purification |
| Other | Strep-Tactin Superflow high capacity 50% suspension | Lucerna-Chem (IBA) | 2-1208-010 | See Methods, CALHM protein purification, Pulldown binding assays |
| Other | Superose 6 10/300 GL | GE Healthcare | 17-5172-01 | See Methods, CALHM protein purification |
