Conformational dynamics of auto-inhibition in the ER calcium sensor STIM1
- Department of Molecular and Cellular Physiology, Stanford University School of Medicine, United States
- Howard Hughes Medical Institute, Stanford University School of Medicine, United States
Figures
Figure 1 with 3 supplements
Parallel orientation of CC2 domains in ctSTIM1 is consistent with the CAD crystal structure.
(A) (top) Schematic overview of domains in ctSTIM1, comprising the membrane-proximal coiled-coil 1 (CC1) domain with helical regions α1 (aa 238–271), α2 (aa 278–304), and α3 (aa 308–337), and the …
Figure 1—figure supplement 1
smFRET histograms for CAD measurements.
Peaks of the histograms are indicated by arrows and represent the predominant ctSTIM1 conformation. The locations of the measurement sites are indicated on a model of ctSTIM1 for each sample. Legend …
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Figure 1—figure supplement 1—source data 1
Time series of donor and acceptor fluorescence and FRET for single molecules.
- https://cdn.elifesciences.org/articles/66194/elife-66194-fig1-figsupp1-data1-v2.xlsx
Figure 1—figure supplement 2
smFRET histograms for CC1 measurements.
Peaks of the histograms are indicated by arrows and represent the predominant ctSTIM1 conformation. The locations of the measurement sites are indicated on a model of ctSTIM1 for each sample. Legend …
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Figure 1—figure supplement 2—source data 1
Time series of donor and acceptor fluorescence and FRET for single molecules.
- https://cdn.elifesciences.org/articles/66194/elife-66194-fig1-figsupp2-data1-v2.xlsx
Figure 1—figure supplement 3
smFRET histograms for CC1:CAD measurements.
Peaks of the histograms are indicated by arrows and represent the predominant ctSTIM1 conformation. The locations of the measurement sites are indicated on a model of ctSTIM1 or CAD for each sample. …
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Figure 1—figure supplement 3—source data 1
Time series of donor and acceptor fluorescence and FRET for single molecules.
- https://cdn.elifesciences.org/articles/66194/elife-66194-fig1-figsupp3-data1-v2.xlsx
Figure 2 with 1 supplement
The apex of CAD in ctSTIM1 deviates from the CAD crystal structure.
(A) Representative smFRET recording at the base of CAD (431:431´) showing stable, high FRET. (B) Ensemble density plots of the initial 2 s of inter-subunit smFRET recordings for sites throughout …
Figure 2—figure supplement 1
CAD apex structure.
(A) Dimerization of ctSTIM1 cysteine mutants by disulfide bond formation in CAD, shown by non-reducing SDS-PAGE. CuP-induced dimerization was virtually absent at basal sites but was evident at all …
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Figure 2—figure supplement 1—source data 1
Raw unedited gel for Figure 2—figure supplement 1A (ctSTIM1 CAD cysteine crosslinking).
- https://cdn.elifesciences.org/articles/66194/elife-66194-fig2-figsupp1-data1-v2.zip
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Figure 2—figure supplement 1—source data 2
Uncropped labeled gel for Figure 2—figure supplement 1A (ctSTIM1 CAD cysteine crosslinking).
- https://cdn.elifesciences.org/articles/66194/elife-66194-fig2-figsupp1-data2-v2.zip
Figure 3 with 1 supplement
The CC1α1 domain is near and parallel to the CC3 domain of the opposing CAD subunit.
(A) Asymmetric inter-subunit smFRET measurements between CC1α1 and CC3 reveal a parallel orientation, with high FRET levels at 242:400´ (top, red) and 274:431´ (bottom, blue) indicating close …
Figure 3—figure supplement 1
CC1α1:CAD dynamics.
(A) Schematic drawing of the measurement sites for inter-subunit smFRET measurements from aa 242 in CC1α1 to sites 400´, 417´, and 431´ in CC3´ (left). An example recording of smFRET transitions at …
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Figure 3—figure supplement 1—source data 1
Time series of FRET, Hidden-Markov Model fits, and state clusters for single molecules.
- https://cdn.elifesciences.org/articles/66194/elife-66194-fig3-figsupp1-data1-v2.xlsx
Figure 4 with 1 supplement
The CC1α2 and CC1α3 domains are closely apposed and directed away from CAD.
(A) High inter-subunit smFRET at the N- and C-termini of the CC1α3 domain (aa 312 and 337, respectively) indicates close parallel apposition of the two helices. The crystal structure of CAD is shown …
Figure 4—figure supplement 1
CC1α3:CC1α3´ dynamics.
(A) Schematic drawing of inter-subunit smFRET measurement sites near the CC1α3 N-terminus. (B) Example traces at aa 307 (top), 309 (center), and 312 (bottom) show large-amplitude fluctuations from …
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Figure 4—figure supplement 1—source data 1
Time series of FRET, Hidden-Markov Model fits, and state clusters for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/66194/elife-66194-fig4-figsupp1-data1-v2.xlsx
Figure 5 with 3 supplements
smFRET-derived models of the configuration of CC1 and CAD in ctSTIM1.
(A) 36 smFRET-derived distances were used to reconstruct the orientations of CC1 domains relative to CAD, yielding two classes of solutions with the CC1α2/α3 domains in a ’stacked' (left) or …
Figure 5—figure supplement 1
CC1-CAD models.
smFRET-constrained modeling produced two different topologies for stacking of the CC1α2/α3 domains. For both classes of solutions, the orientation of CC1α1 parallel to CC3 on the adjacent subunit …
Figure 5—figure supplement 2
Mass spectrometry analysis of ctSTIM1 crosslinking by BS3.
(A) SDS-PAGE of wild-type and mutant ctSTIM1 treated with BS3 to induce lysine-lysine crosslinking. Wild-type ctSTIM1 was treated with BS3-d0 as a reference, and four different ctSTIM1 variants were …
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Figure 5—figure supplement 2—source data 1
Raw unedited gel for Figure 5—figure supplement 2A (ctSTIM1 BS3 crosslinking).
- https://cdn.elifesciences.org/articles/66194/elife-66194-fig5-figsupp2-data1-v2.zip
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Figure 5—figure supplement 2—source data 2
Uncropped labeled gel for Figure 5—figure supplement 2A (ctSTIM1 BS3 crosslinking).
- https://cdn.elifesciences.org/articles/66194/elife-66194-fig5-figsupp2-data2-v2.zip
Figure 5—figure supplement 3
Mass spectrometry analysis of ctSTIM1 crosslinking by EDC.
(A) SDS-PAGE analysis of ctSTIM1 treated with zero-length amine-carboxyl crosslinker EDC. Monomer (blue) and dimer (red) bands were cut from the gel for analysis by mass spectrometry. For the …
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Figure 5—figure supplement 3—source data 1
Raw unedited gel for Figure 5—figure supplement 3A (ctSTIM1 EDC crosslinking).
- https://cdn.elifesciences.org/articles/66194/elife-66194-fig5-figsupp3-data1-v2.zip
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Figure 5—figure supplement 3—source data 2
Uncropped labeled gel for Figure 5—figure supplement 3A (ctSTIM1 EDC crosslinking).
- https://cdn.elifesciences.org/articles/66194/elife-66194-fig5-figsupp3-data2-v2.zip
Figure 6 with 1 supplement
The Stormorken R304W mutation releases CC1 from CAD.
(A) The R304W mutation reduced inter-subunit smFRET between aa 242 on CC1α1 and sites 400´, 417´, and 431´ on CC3´, indicating separation of CC1α1 from CAD. The mutation also reduced intra-subunit …
Figure 6—figure supplement 1
Orai1-mediated Ca2+ influx evoked by ctSTIM1 fragments.
HEK293 cells expressing the indicated ctSTIM1 variant and Orai1-GFP were exposed sequentially to 2 mM Ca2+, 0 Ca2+, and 2 mM Ca2+. The fura-2 350/380 fluorescence ratio is shown for ctSTIM1 (aa …
Figure 7 with 2 supplements
Close pairing of CC1 domains along their entire length in the activated state of full-length STIM1 (flSTIM1) in vivo.
(A) Western-blot analysis of diamide-induced cysteine crosslinking of flSTIM1-WT, flSTIM1-A268C, flSTIM1-T307C and flSTIM1-S339C in HEK293 cells, under resting (2 mM Ca2+) or store-depleted (0 mM Ca2…
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Figure 7—source data 1
Raw unedited and uncropped labeled western blots for Figure 7A (WT).
- https://cdn.elifesciences.org/articles/66194/elife-66194-fig7-data1-v2.zip
Figure 7—figure supplement 1
CC1 cysteine crosslinking in ctSTIM1 and flSTIM1.
(A) Cysteine crosslinking throughout the CC1 domain of ctSTIM1 mutants in vitro produced by copper phenanthroline and detected by non-reducing SDS-PAGE (see Materials and methods). Crosslinking …
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Figure 7—figure supplement 1—source data 1
Raw unedited and uncropped labeled gel and western blots for Figure 7—figure supplement 1.
- https://cdn.elifesciences.org/articles/66194/elife-66194-fig7-figsupp1-data1-v2.zip
Figure 7—figure supplement 2
Mass spectrometry analysis of BS3 crosslinking of active ctSTIM1 mutants.
Mass-spectrometry analysis of ctSTIM1 variants 2LS (L248S + L251S, B), R304W (C), and T307C (D) showed similarly altered link patterns relative to WT (A). Crosslinked residue pairs for each sample …
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Figure 7—figure supplement 2—source data 1
List of crosslinked residues from mass spectrometry.
- https://cdn.elifesciences.org/articles/66194/elife-66194-fig7-figsupp2-data1-v2.xlsx
Figure 8
Possible conformational trajectories of flSTIM1 activation in vivo.
(A) In the resting state, the parallel orientation of CC1α1 and CC3 domains implies that CAD is held with its apex close to and pointed towards the ER membrane, effectively shielding critical …
Author response image 1
Dimers of flSTIM1 show anomalously low mobility in SDS-PAGE.
Purified flSTIM1 was treated with 0.1 mM diamide for 10 min. In lane 2, 10 mM TCEP (reducing agent) was added to the crosslinked sample 10 min before running the gel. Coomassie blue SDS-PAGE with MW …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (human) | STIM1 | Origene | ||
| Strain, strain background (Escherichia coli) | BL21 | New England Biolabs | C25271 | |
| Strain, strain background (Escherichia coli) | CVB101 | Avidity | CVB101 | |
| Cell line (human) | HEK293 | ATCC | CRL-1573 | |
| Antibody | Mouse monoclonal anti-mCherry | Takara Bio | 632,543RRID:AB_2307319 | (1:2000) |
| Antibody | Donkey polyclonal anti-mouse IgG | LI-COR | 926–032212RRID:AB_621847 | (1:10000) |
| Recombinant DNA reagent | pAC6 (vector) | Avidity | ||
| Recombinant DNA reagent | mCherry-labeled STIM1 (plasmid) | doi:10.1083/jcb.200604014 | ||
| Recombinant DNA reagent | Orai1-GFP (plasmid) | doi:10.1074/jbc.M703573200 | ||
| Peptide, recombinant protein | TEV protease | MCLAB | TEV-200 | |
| Peptide, recombinant protein | BSA-biotin | Sigma Aldrich | A8549 | |
| Peptide, recombinant protein | Neutravidin | Thermo Fisher | 31000 | |
| Peptide, recombinant protein | Glucose oxidase | Sigma Aldrich | G2133 | |
| Peptide, recombinant protein | Catalase | Sigma Aldrich | C9322 | |
| Peptide, recombinant protein | Trypsin/LysC protease | Promega | V5071 | |
| Commercial assay or kit | QuikChange II | Agilent | 200524 | |
| Commercial assay or kit | PEG/PEG-biotin | Laysan Bio | BIO-PEG-SVA-5K & MPEG-SVA-5K | |
| Chemical compound, drug | TCEP | Thermo Fisher Scientific | 77720 | |
| Chemical compound, drug | Alexa Fluor 555 | Invitrogen Life Technologies | A-20346 | |
| Chemical compound, drug | Alexa Fluor 647 | Invitrogen Life Technologies | A-20347 | |
| Chemical compound, drug | Biotinylated lipids 18:1 Biotinyl Cap PE | Avanti Polar Lipids | 870273 C | |
| Chemical compound, drug | egg-PC lipids | Avanti Polar Lipids | 840051 C | |
| Chemical compound, drug | Cyclooctatetraene | Sigma Aldrich | 138924 | |
| Chemical compound, drug | BS3-d0 | Sigma Aldrich | 21590 | |
| Chemical compound, drug | BS3-d4 | Sigma Aldrich | 21595 | |
| Chemical compound, drug | EDC | Thermo Fisher Scientific | 77149 | |
| Chemical compound, drug | Sulfo-NHS | Thermo Fisher Scientific | 24520 | |
| Chemical compound, drug | ProteaseMax | Promega | V2071 | |
| Chemical compound, drug | Cyclopiazonic acid (CPA) | Sigma Aldrich | C1530 | |
| Chemical compound, drug | Protease inhibitor cocktail | Cell Signaling Technology | 5871 S | |
| Chemical compound, drug | Lipofectamine 2000 | Thermo Scientific | 11668027 | |
| Chemical compound, drug | Poly-D-lysine | Sigma Aldrich | P-7405 | |
| Chemical compound, drug | fura-2/AM | Invitrogen | F-1221 | |
| Software, algorithm | μManager | doi:https://doi.org/10.1002/0471142727 | RRID:SCR_016865 | https://micro-manager.org/ |
| Software, algorithm | SMART: Single Molecule Analysis Research Tool | doi:10.1371/journal.pone.0030024 | https://simtk.org/projects/smart/ | |
| Software, algorithm | Crystallography and NMR System (CNS) | doi:10.1038/nprot.2007.406; doi:https://doi.org/10.1107/S0907444998003254 | RRID:SCR_014223 | http://cns-online.org/v1.2/ |
| Software, algorithm | PyMOL Molecular Graphics System ver. 1.8 | Schrödinger, LLC | RRID:SCR_000305 | http://www.pymol.org/ |
| Software, algorithm | ProDy | doi:10.1093/bioinformatics/btr168 | http://prody.csb.pitt.edu/ | |
| Software, algorithm | Flexpepdock server of Rosetta | doi:10.1093/nar/gkr431 | Rosetta, RRID:SCR_015701 | http://flexpepdock.furmanlab.cs.huji.ac.il/ |
| Software, algorithm | Byonic v2.12.0 or v2.14.27 | Protein Metrics | RRID:SCR_016735 | |
| Other | NiNTA beads | Qiagen | 30210 | |
| Other | SnakeSkin | Life Technologies | 68700 | |
| Other | HiTrap Q column | GE Healthcare Life Sciences | 17-1153-01 | |
| Other | Quartz microscope slides | G. Finkenbeiner Inc. | For smFRET | |
| Other | Microscope coverslips | Erie Scientific | 24 × 40 1.5 001 | For smFRET |
| Other | Liposome Extruder Set | Avanti Polar Lipids | 610023 | |
| Other | PC Membranes 0.1 µm | Avanti Polar Lipids | 610005-1EA | |
| Other | Sepharose CL-4B column | Sigma Aldrich | CL4B200 | |
| Other | OBIS 532 nm LS 150 mW laser | Coherent | 1280719 | For smFRET |
| Other | OBIS 637 nm LX 140 mW laser | Coherent | 1196626 | For smFRET |
| Other | Dichroic beamsplitter | Semrock | FF652-Di01−25 × 36 | For smFRET |
| Other | Donor band-pass filter | Semrock | FF01-580/60-25-D | For smFRET |
| Other | Acceptor band-pass filter | Semrock | FF01-731/137-25 | For smFRET |
| Other | Emission beam splitter | Cairn Research | OptoSplit-II | For smFRET |
| Other | EM-CCD camera | Andor | iXon DU897E | For smFRET |
| Other | Polychrome II | TILL Photonics | For calcium imaging | |
| Other | emission filter | Semrock | FF02-534/30-25 | For calcium imaging |
| Other | emission filter >480 nm | Chroma Technology Corp. | For calcium imaging | |
| Other | Flash4.0 sCMOS camera | Hamamatsu Corp. | For calcium imaging |
Additional files
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Supplementary file 1
smFRET-derived distance measurements used to construct the CC1-CAD model.
The table lists the complete set of amino acid pairs, their predominant smFRET efficiencies, the corresponding smFRET-derived distances, and the range of distance values used for generating the models shown in Figure 5A and Figure 5—figure supplement 1. The 'FRET peak' and 'Distance' values correspond to the peaks of the smFRET histograms and the associated calculated distances shown in Figure 1—figure supplements 1–3. For sites with both liposome and avitag measurements, the liposome measurement was used to constrain the CC1:CAD model. The ‘Range’ values indicate the allowable distance bounds for each pair of modeled residues (see Materials and methods).
- https://cdn.elifesciences.org/articles/66194/elife-66194-supp1-v2.docx
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Supplementary file 2
PyMOL file containing the stacked CC1-CAD models shown in Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/66194/elife-66194-supp2-v2.pse
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Supplementary file 3
PyMOL file containing the wedged CC1-CAD models shown in Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/66194/elife-66194-supp3-v2.pse
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Transparent reporting form
- https://cdn.elifesciences.org/articles/66194/elife-66194-transrepform1-v2.docx
