Cryo-EM structures of the caspase-activated protein XKR9 involved in apoptotic lipid scrambling
- Department of Biochemistry, University of Zurich, Switzerland
Figures
Figure 1 with 3 supplements
Biochemical and functional properties of rXKR9.
Size-exclusion chromatogram of full-length (A) and caspase-treated (B) rXKR9. Insets show SDS–PAGE gel of the pooled and concentrated peak sample. (C) SDS–PAGE gel of rXKR9 extracted from different …
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Figure 1—source data 1
SDS–PAGE gel of full-length rXKR9 shown in Figure 1A.
- https://cdn.elifesciences.org/articles/69800/elife-69800-fig1-data1-v2.zip
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Figure 1—source data 2
SDS–PAGE gel of caspase-3 treated rXKR9 shown in Figure 1B.
- https://cdn.elifesciences.org/articles/69800/elife-69800-fig1-data2-v2.zip
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Figure 1—source data 3
SDS–PAGE gel of full-length or caspase-3-treated rXKR9 extracted from respective proteoliposomes shown in Figure 1C.
- https://cdn.elifesciences.org/articles/69800/elife-69800-fig1-data3-v2.zip
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Figure 1—source data 4
Normalized individual traces of scrambling experiments displayed in Figure 1D.
- https://cdn.elifesciences.org/articles/69800/elife-69800-fig1-data4-v2.zip
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Figure 1—source data 5
SDS–PAGE gel of the XKR8/basigin complex shown in Figure 1E.
- https://cdn.elifesciences.org/articles/69800/elife-69800-fig1-data5-v2.zip
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Figure 1—source data 6
Silver-stained SDS–PAGE gel of the XKR9 co-expressed with basigin ahown in Figure 1G.
- https://cdn.elifesciences.org/articles/69800/elife-69800-fig1-data6-v2.zip
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Figure 1—source data 7
In-gel fluorescence of Venus-tagged proteins detected in SDS–PAGE gel of the XKR8 or XKR9 expressed with basigin shown in Figure 1G.
- https://cdn.elifesciences.org/articles/69800/elife-69800-fig1-data7-v2.zip
Figure 1—figure supplement 1
Biochemical characterization of rXKR9.
(A) Multi-angle light scattering coupled to size-exclusion chromatography of detergent-solubilized rXKR9. Continuous red trace corresponds to the absorption at 280 nm. The corrected molecular …
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Figure 1—figure supplement 1—source data 1
Normalized individual traces of scrambling experiments displayed in Figure 1—figure supplement 1C.
- https://cdn.elifesciences.org/articles/69800/elife-69800-fig1-figsupp1-data1-v2.zip
Figure 1—figure supplement 2
Preparation of rXKR9-Sb1XKR9 complexes.
(A) Sequence of the sybody Sb1XKR9. Secondary structure and randomized complementary determining regions are indicated. Residues in contact with rXKR9 are highlighted in cyan. Size-exclusion …
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Figure 1—figure supplement 2—source data 1
SDS–PAGE gel of purified full-length rXKR9 in complex with Sb1XKR9 shown in Figure 1B.
- https://cdn.elifesciences.org/articles/69800/elife-69800-fig1-figsupp2-data1-v2.zip
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Figure 1—figure supplement 2—source data 2
SDS–PAGE gel of purified caspase-3 treated rXKR9 in complex with Sb1XKR9 shown in Figure 1B.
- https://cdn.elifesciences.org/articles/69800/elife-69800-fig1-figsupp2-data2-v2.zip
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Figure 1—figure supplement 2—source data 3
Mass spectrum of caspase-3-treated XKR9 shown in Figure 1—figure supplement 2D.
- https://cdn.elifesciences.org/articles/69800/elife-69800-fig1-figsupp2-data3-v2.zip
Figure 1—figure supplement 3
Low-resolution cryo-EM reconstruction of a hXKR8-basigin complex.
(A) Representative cryo-EM micrograph acquired with a Tecnai G2 Polara microscope and a K2 summit camera. (B) 2D class averages of a hXKR8-basigin complex. (C) Data processing workflow. After …
Figure 2 with 4 supplements
Cryo-EM maps of rXKR9-Sb1XKR9 complexes.
(A) Cryo-EM density of complexes of the full-length construct at 3.66 Å (contoured at 9.5 σ) and (B), the caspase-treated protein at 4.3 Å (contoured at 8.8 σ). The relationship between orientations …
Figure 2—figure supplement 1
Cryo-EM reconstruction of the full-length rXKR9-Sb1XKR9 complex.
(A) Representative micrograph of the complex acquired with a Titan Krios G3i microscope equipped with a K3 camera. (B) 2D class averages of the rXKR9-Sb1XKR9 dataset. (C) Data processing workflow. …
Figure 2—figure supplement 2
Cryo-EM reconstruction of the caspase-treated rXKR9-Sb1XKR9 complex.
(A) Representative micrograph of the complex acquired with a Titan Krios G3i microscope equipped with a K3 camera. (B) 2D class averages of the caspase-treated rXKR9-Sb1XKR9 dataset. (C) Data …
Figure 2—figure supplement 3
Model validation.
FSC plots of atomic rXKR9-Sb1XKR9 models refined against the respective cryo-EM data. (A) Full-length rXKR9-Sb1XKR9, (B) Caspase-3-treated rXKR9-Sb1XKR9. FSCsum (magenta) is calculated for the full …
Figure 2—figure supplement 4
Cryo-EM density.
(A) Density of the detergent belt (magenta) in the ab initio cryo-EM reconstruction of the full-length rXKR9-Sb1XKR9 complex (contoured at 5.8σ). Relationships between orientations are indicated. (B)…
Figure 3 with 1 supplement
XKR9-SB1XKR9 complex.
Ribbon representation of Sb1XKR9 complexes of (A, B) full-length and (C, D) caspase-treated rXKR9. The relationship between orientations is indicated. rXKR9 is shown in unique colors, Sb1XKR9 in …
Figure 3—figure supplement 1
rXKR9-Sb1XKR9 interactions.
(A) Ribbon representation of the rXKR9-Sb1XKR9 interaction interface. Color code is as in Figure 3A. Secondary structure elements and complementary determining regions are indicated. (B) Stick model …
Figure 4 with 1 supplement
Sequence and topology.
Sequence alignment of human XKR8 (hXKR8, NCBI: NP_060523.2) and rXKR9 (NCBI: NP_001012229.1), secondary structure elements are indicated. Identical residues are highlighted in green, similar …
Figure 4—figure supplement 1
Hydropathy analysis of the rXKR9 sequence.
(A) Prediction of membrane-spanning helices (numbered) by the TMHMM server (Krogh et al., 2001). (B) rXKR9 sequence comparing location of predicted (top, magenta) and actual TM helices (bottom …
Figure 5 with 2 supplements
Features of the rXKR9 structure of potential functional relevance.
(A–D) Cavities on the surface of the rXKR9 structure that are suitable for lipid interactions. Segments of the protein are displayed as ribbon. Sections of the molecular surface are shown and …
Figure 5—figure supplement 1
Lipid density.
(A) Residual density in the cavities C1 and C2 at the domain interface of rXKR9. Densities in C1 (left, 1) and C2 (right, 2) were interpreted by phosphatidylcholine molecules, another density in C2 …
Figure 5—figure supplement 2
Surface electrostatics.
(A) Ribbon representation of different orientations of rXKR9 viewed from within the membrane (top) and electrostatic potential mapped on the molecular surface of rXKR9 (bottom). (B) Ribbon …
Figure 6
Binding site of the C-terminus.
(A) Binding of the C-terminus of rXKR9 to a cleft of the protein. rXKR9 excluding its C-terminus is shown as surface representation and colored as in Figure 4D. The C-terminal peptide (CT) defined …
Author response image 1
Lipid scrambling assay of the XKR8-basigin complex.
(A) Size exclusion chromatogram of proteins extracted from proteoliposomes containing a full-length reconstituted XKR8-basigin complex (uncleaved), the same proteoliposomes treated with caspase-3 …
Author response image 2
SDS PAGE gel showing mass differences between full-length and caspase-cleaved samples.
Left, SDS PAGE gel of full-length and caspase-3 treated samples with the integrated lanes numbered and indicated by yellow boxes. Right, integrated density of the gel along the direction of …
Tables
Table 1
Cryo-EM data collection, refinement, and validation statistics.
| Dataset 1 rXkr9 (EMDB-13155) (PDB 7P14) | Dataset 2 rXkr9 caspase −3 cleaved (EMDB-13157) (PDB 7P16) | Dataset 3 hXkr8+hBSG | |
|---|---|---|---|
| Data collection and processing | |||
| Microscope | FEI Titan Krios | FEI Titan Krios | Polara |
| Camera | Gatan K3 GIF | Gatan K3 GIF | Gatan K2 |
| Magnification | 130,000 | 130,000 | 130,000 |
| Voltage (kV) | 300 | 300 | 300 |
| Electron exposure (e–/Å2) | 70 | 70 | 35 |
| Defocus range (μm) | −1.0 to −2.4 | −1.0 to −2.4 | −0.8 to −2.5 |
| Pixel size * (Å) | 0.651 (0.3255) | 0.651 (0.3255) | 1.34 |
| Initial number of micrographs (no.) | 12,396 | 14,929 | 2212 |
| Initial particle images (no.) | 6,252,082 | 8,302,932 | 465,373 |
| Final particle images (no.) | 866,439 | 444,358 | 54,519 |
| Symmetry imposed | C1 | C1 | C1 |
| Map resolution (Å) FSC threshold | 3.66 0.143 | 4.3 0.143 | 15.3 0.143 |
| Map resolution range (Å) | 3.0–6.0 | 3.7–7.0 | |
| Refinement | |||
| Model resolution (Å) FSC threshold | 3.7 0.5 | 4.3 0.5 | |
| Map sharpening b-factor (Å2) | −199.6 | −229.9 | |
| Model vs Map CC (mask) | 0.76 | 0.74 | |
| Model composition Non-hydrogen atoms Protein residues Ligand | 3796 456 PLC, P5S | 3621 445 PLC | |
| B factors (Å2) Protein Ligand | 53.43 47.62 | 68.75 49.33 | |
| R.m.s. deviations Bond lengths (Å) Bond angles (°) | 0.003 0.533 | 0.002 0.453 | |
| Validation MolProbity score Clashscore Poor rotamers (%) | 1.54 7.45 0.00 | 1.51 7.29 0.00 | |
| Ramachandran plot Favored (%) Allowed (%) Disallowed (%) | 97.3 2.7 0.00 | 97.48 2.52 0.00 |
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*Values in parentheses indicate the pixel size in super-resolution.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (human) | HEK239T | ATCC | CRL-3216 | RRID:CVCL_0063 |
| Cell line (human) | HEK293S GnTI- | ATCC | CRL-3022 | RRID:CVCL_A785 |
| Cell line (human) | mTMEM16F HEK293T | Alvadia et al., 2019 | NA | |
| Chemical compound, drug | 18:1 06:0 NBD-PE | Avanti Polar Lipids | 810155C | |
| Chemical compound, drug | 18:1 06:0 NBD-PS | Avanti Polar Lipids | 810194C | |
| Chemical compound, drug | 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'rac-glycerol) (18:1 06:0 POPG) | Avanti Polar Lipids | 840457C | |
| Chemical compound, drug | 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:1 06:0 POPC) | Avanti Polar Lipids | 850457C | |
| Chemical compound, drug | 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (18:1 06:0 POPE) | Avanti Polar Lipids | 850757C | |
| Chemical compound, drug | Benzamidine | Sigma | B6506 | |
| Chemical compound, drug | Biotin | Sigma | B4501 | |
| Chemical compound, drug | Calcium nitrate tetrahydrate | Sigma | C4955 | |
| Chemical compound, drug | CHAPS | Sigma | C3023 | |
| Chemical compound, drug | Chloramphenicol | Sigma | C1919 | |
| Chemical compound, drug | Chloroform | Fluka | 25690 | |
| Chemical compound, drug | Cholesterol | Sigma | C3045 | |
| Chemical compound, drug | cOmplete, EDTA-free Protease Inhibitor Cocktail | Roche | 5056489001 | |
| Chemical compound, drug | D-Desthiobiotin | Sigma | D1411 | |
| Chemical compound, drug | DDM | Anatrace | D310S | |
| Chemical compound, drug | Diethyl ether | Sigma | 296082 | |
| Chemical compound, drug | Digitonin High Purity | Merck Millipore | 300410 | |
| Chemical compound, drug | DNase I | AppliChem | A3778 | |
| Chemical compound, drug | DTT | ThermoFisher Scientific | R0861 | |
| Chemical compound, drug | Dulbecco's modified Eagle's medium - high glucose | Gibco, ThermoFisher Scientific | 41966029 | |
| Chemical compound, drug | Etyleneglycol - bis (2 - aminoethylether) - N,N,N′,N′- tetraacetic acid | Sigma | E3889 | |
| Chemical compound, drug | Fetal bovine serum | Sigma | F7524 | |
| Chemical compound, drug | GDN | Anatrace | GDN101 | |
| Chemical compound, drug | Glycerol 99% | Sigma | G7757 | |
| Chemical compound, drug | HCl | Merck 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 | Lauryl-maltose-neopentyl glycol (LMNG) | Anatrace | NG310 | |
| Chemical compound, drug | Leupeptin | AppliChem | A2183 | |
| Chemical compound, drug | L-glutamine | Sigma | G7513 | |
| Chemical compound, drug | Penicillin–streptomycin | Sigma | P0781 | |
| Chemical compound, drug | Pepstatin | AppliChem | A2205 | |
| Chemical compound, drug | Phosphate buffered saline | Sigma | D8537 | |
| Chemical compound, drug | PMSF | Sigma | P7626 | |
| Chemical compound, drug | Polyethyleneimine MAX 40 kDa | PolySciences Inc | 24765–1 | |
| Chemical compound, drug | Potassium chloride | Sigma | 746346 | |
| Chemical compound, drug | Sodium chloride | Sigma | 71380 | |
| Chemical compound, drug | Sodium dithionite (Sodium hydrosulfite) | Sigma | 157953 | |
| Chemical compound, drug | Soybean Polar Lipids | Avanti Polar Lipids | 541602C | |
| Chemical compound, drug | TCEP | Hampton | HR2-651 | |
| Chemical compound, drug | Terrific broth | Sigma | T9179 | |
| Chemical compound, drug | Tetracycline hydrochloride | Sigma | T7660 | |
| Chemical compound, drug | Tris | AppliChem | A1379 | |
| Chemical compound, drug | Valproic acid | Sigma | P4543 | |
| Commercial assay or kit | 4–20% Mini-PROTEAN TGX Precast Protein Gels, 15-well, 15 µl | BioRad Laboratories | 4561096DC | |
| Commercial assay or kit | Amicon Ultra-4 Centrifugal Filters Ultracel 10K, 4 ml | Merck Millipore | UFC801096 | |
| Commercial assay or kit | Amicon Ultra-4 Centrifugal Filters Ultracel 50K, 4 ml | Merck Millipore | UFC805096 | |
| Commercial assay or kit | Amicon Ultra-4 Centrifugal Filters Ultracel 100K, 4 ml | Merck Millipore | UFC810024 | |
| Commercial assay or kit | Biobeads SM-2 adsorbents | BioRad Laboratories | 152–3920 | |
| Commercial assay or kit | EZ-link NHS-PEG4-biotin | ThermoFisher Scientific | A39259 | |
| Commercial assay or kit | MiniExtruder | Avanti Polar Lipids | 610023 | |
| Commercial assay or kit | Ni-NTA resin | ABT Agarose Bead Technologies | 6BCL-NTANi-X | |
| Commercial assay or kit | Nunc 384-well plate MaxiSorp | ThermoFisher Scientific | 460372 | |
| Commercial assay or kit | PC membrane 0.2 μm | Avanti Polar Lipids | 610006 | |
| Commercial assay or kit | PD-10 desalting column | Sigma | GE17-0851-01 | |
| Commercial assay or kit | Pierce Streptavidin Plus UltraLink Resin | ThermoFisher Scientific | 53117 | |
| Commercial assay or kit | QuantiFoil R1.2/1.3 Au 200 mesh | Electron Microscopy Sciences | Q2100AR1.3 | |
| Commercial assay or kit | Silver Stain Plus | BioRad Laboratories | 161–0449 | |
| Commercial assay or kit | SRT-10C SEC 100 | Sepax Technologies | 239100–10030 | |
| Commercial assay or kit | Strep-Tactin Superflow high capacity 50% suspension | IBA LifeSciences | 2-1208-010 | |
| Commercial assay or kit | Superdex 200 10/300 GL | Cytiva | 17517501 | |
| Commercial assay or kit | Superdex 200 Increase 3.2/300 | Cytiva | 28990946 | |
| Commercial assay or kit | Superdex 200 Increase 5/150 GL | Cytiva | 28990945 | |
| Commercial assay or kit | Superdex 75 10/300 GL | Cytiva | 17517401 | |
| Commercial assay or kit | Superose 6 10/300 GL | Cytiva | 17517201 | |
| Commercial assay or kit | Superose 6 Increase 5/150 | Cytiva | 29091597 | |
| Commercial assay or kit | Ultrafree MC GV 0.22 μm centrifugal filter | Merck Millipore | UFC30GVNB | |
| Commercial assay or kit | Ultrafree - MC -HV, DURAPORE PVDF 0.1 μm | Merck Millipore | UFC30VV00 | |
| Other | BioQuantum Energy Filter | Gatan | NA | |
| Other | Fluoromax spectrometer | Horiba | NA | |
| Other | HPL6 | Maximator | NA | |
| Other | K2 Summit Direct Detector | Gatan | NA | |
| Other | K3 Summit Direct Detector | Gatan | NA | |
| Other | qPCR machine Mx3005p | Agilent | NA | |
| Other | Sybody library | Generous gift from the Seeger laboratory | NA | |
| Other | Tecnai G2 Polara | ThermoFisher Scientific | NA | |
| Other | Titan Krios G3i | ThermoFisher Scientific | NA | |
| Other | UT-rEX Refractometer | Wyatt Technology | NA | |
| Other | Viber Fusion FX7 imaging system | Witec | NA | |
| Other | Vitrobot Mark IV | ThermoFisher Scientific | NA | |
| Other | μDAWN MALS Detector | Wyatt Technology | NA | |
| Recombinant DNA reagent | human Basigin open reading frame | GenScript | HUMBSG | |
| Recombinant DNA reagent | human Xkr8 open reading frame | GenScript | NM_018053 | |
| Recombinant DNA reagent | Mammalian expression vector with C-terminal 3C cleavage site, GFP-tag and myc-tag | Dutzler laboratory | NA | |
| Recombinant DNA reagent | Mammalian expression vector with N-terminal streptavidin binding peptide, myc-tag and 3C cleavage site | Dutzler laboratory | NA | |
| Recombinant DNA reagent | Mammalian expression vector with N-terminal streptavidin binding peptide, myc-tag, Venus-tag and 3C cleavage site | Dutzler laboratory | NA | |
| Recombinant DNA reagent | Mouse mTMEM16F open reading frame | Dharmacon - Horizon Discovery | GenBank#BC060732 | |
| Recombinant DNA reagent | pSb_init | Generous gift from the Seeger laboratory | RRID:addgene_110100 | |
| Recombinant DNA reagent | rat basigin open reading frame | GenScript | NM_012783 | |
| Recombinant DNA reagent | rat XKR9 open reading frame | GenScript | NM_001012229 | |
| Peptide, recombinant protein | HRV 3C protease | Expressed (pET_3C) and purified in Dutzler laboratory | NA | |
| Peptide, recombinant protein | Human Caspase-3, recombinant | BioVision | 1083 | |
| Peptide, recombinant protein | Streptavidin | Expressed and purified in Dutzler laboratory | NA | |
| Software, algorithm | 3DFSC | Tan et al., 2017 | https://3dfsc.salk.edu/ | |
| Software, algorithm | ASTRA7.2 | Wyatt Technology | https://www.wyatt.com/products/software/astra.html | RRID:SCR_016255 |
| Software, algorithm | Chimera v.1.15 | Pettersen et al., 2004 | https://www.cgl.ucsf.edu/chimera/ | RRID:SCR_004097 |
| Software, algorithm | ChimeraX v.1.1.1 | Pettersen et al., 2021 | https://www.rbvi.ucsf.edu/chimerax/ | RRID:SCR_015872 |
| Software, algorithm | Coot v.0.9.4 | Emsley and Cowtan, 2004 | https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ | RRID:SCR_014222 |
| Software, algorithm | cryoSPARC v.3.0.1/v.3.2.0 | Structura Biotechnology Inc | https://cryosparc.com/ | RRID:SCR_016501 |
| Software, algorithm | DINO | http://www.dino3d.org | RRID:SCR_013497 | |
| Software, algorithm | EPU2.9 | ThermoFisher Scientific | NA | |
| Software, algorithm | Codon optimization tool | Integrated DNA technologies | https://eu.idtdna.com/pages/tools/codon-optimization-tool | |
| Software, algorithm | Phenix | Liebschner et al., 2019 | https://www.phenix-online.org/ | RRID:SCR_014224 |
| Software, algorithm | RELION 3.0.7 | Zivanov et al., 2018 | https://www3.mrc-lmb.cam.ac.uk/relion/ | RRID:SCR_016274 |
| Software, algorithm | SerialEM3.7 | Mastronarde, 2005 | https://bio3d.colorado.edu/SerialEM/ | |
| Software, algorithm | WEBMAXC calculator | Bers et al., 2010 | https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/ maxchelator/webmaxc/webmaxcS.htm | RRID:SCR_018807 |
| Strain | E. coli MC1061 | ThermoFisher Scientific | C66303 |
Additional files
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Source data 1
Cumulated source data.
- https://cdn.elifesciences.org/articles/69800/elife-69800-data1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/69800/elife-69800-transrepform-v2.docx
