Coiled-Coil Formation Conveys a STIM1 Signal from ER Lumen to Cytoplasm

Abstract

STIM1 and STIM2 are endoplasmic reticulum (ER) membrane proteins that sense decreases in ER-luminal free Ca²⁺ and, through a conformational change in the STIM cytoplasmic domain, control gating of the plasma membrane Ca²⁺ channel ORAI1. To determine how STIM1 conveys a signal from the ER lumen to the cytoplasm, we studied the Ca²⁺-dependent conformational change of engineered STIM1 proteins in isolated ER membranes and, in parallel, physiological activation of these proteins in cells. We find that conserved "sentinel" features of the CC1 region help to prevent activation while Ca²⁺ is bound to STIM ER-luminal domains. Reduced ER-luminal Ca²⁺ drives a concerted conformational change, in which STIM luminal domains rearrange and the STIM transmembrane helices and initial parts of the CC1 regions pair in an extended coiled coil. This intradimer rearrangement overcomes the relatively weak CC1-SOAR/CAD interactions that hold STIM in an inactive conformation, releasing the SOAR/CAD domain to activate ORAI channels.

Keywords: ER-plasma membrane junction; STIM1; STIM2; calcium imaging; coiled coil; conformational change; disulfide crosslinking; evolution; store-operated calcium entry; transmembrane.

Figure 1. Structural hypotheses on STIM1 activation tested in this work

A, Cartoon of the inactive STIM1 dimer. The EF-SAM domains comprise residues 58–201 of each monomer; the transmembrane (TM) helices, residues 214–232; the CC1 regions, residues 233–343; and SOAR/CAD, residues 344–442. The CC1-SOAR/CAD interface of inactive STIM1 is portrayed as spanning CC1 residues L248–L261, based on literature cited in the text. The cartoon depicts one possible configuration of inactive STIM1, in which the Ca²⁺-bound STIM1 EF-SAM domains do not interact and the transmembrane helices are spatially separated. An alternative configuration in which the luminal domains in cells are stabilized in close proximity, but the transmembrane helices are held apart, would yield similar data in our experimental tests. B, Cartoon of the hypothesized active state of STIM1. The defining feature of active STIM1 is the release of SOAR/CAD from CC1. It has been proposed that SOAR/CAD release is triggered by burial of L251 and surrounding residues of the CC1-SOAR/CAD interface in the core of a CC1-CC1 coiled coil (Zhou et al., 2013). C, The three hypotheses tested— Upper, that the TM helices come together upon activation; Middle, that activation coincides with the formation of a coiled-coil in the region of CC1 containing L251; Lower, that activation entails helical apposition along the entire segment connecting the TM helices and the CC1-SOAR/CAD interface surrounding L251. The STIM1 mutants examined and their phenotypes are summarized in Table S1.

Figure 2. Disulfide crosslinking to assess packing of STIM1 transmembrane helices

A, Confocal micrographs of HeLa cells expressing GFP-tagged wild-type STIM1 or GFP-tagged cysteine-less STIM1, as indicated, at rest (Before TG) and after depleting ER Ca²⁺ stores by treatment with 1 µM thapsigargin (After TG). Scale bars, 10 µm. B, Western blots showing crosslinking of the specified single-cysteine mutants of STIM1 in isolated cellular membranes incubated in the presence of EGTA or Ca²⁺. The samples were subjected to nonreducing SDS-polyacrylamide gel electrophoresis, and the blots were probed with anti-STIM1 antibody. Crosslinked STIM1 dimer is marked by the upper arrow, and STIM1 monomer by the lower arrow. C, Crosslinking efficiencies at STIM1 residues 214–234, defined as the percentage of STIM1 in the crosslinked dimer band, in the presence of EGTA (blue) or Ca²⁺ (magenta). Peaks of the superposed curve define one face of the transmembrane helix. Data from three biological replicates. Error bars report SEM. D, Western blot showing the effect of the activating mutation D76A on crosslinking of STIM1^216C and STIM1^219C, in the presence of EGTA or Ca²⁺. See also Figure S1.

Figure 3. Crosslinking at A230C as a function of Ca²⁺ concentration

A, Confocal micrographs of HeLa cells expressing GFP-STIM1(A230C), at rest (−) and after store depletion with 1 µM thapsigargin (TG), documenting the response of the A230C protein in cells. Scale bar, 10 µm. B, Crosslinking of STIM1(A230C) in isolated membranes incubated at the specified Ca²⁺ concentrations, assessed as in Figure 2. C, Efficiency of STIM-STIM dimer formation at each Ca²⁺ concentration is plotted for four biological replicates.

Figure 4. Involvement of CC1 residues 246–252 in a Ca²⁺-dependent conformational switch

A, Crosslinking of STIM1 with single cysteine replacements at residues 250–252, as indicated, assessed as in Figure 2. Representative of three biological replicates. Control samples incubated without addition of iodine were included to determine background crosslinking. The STIM1 dimer band is marked with an arrow. B,C, Confocal micrographs of HeLa cells expressing GFP-STIM1(L251C) (B) or GFP-STIM1(L251S) (C) at rest (−) and after store depletion (TG). Scale bars, 10 µm. D, Effect of the activating mutation L251S on crosslinking of STIM1(A230C), in the presence of EGTA or Ca²⁺. E, Crosslinking of STIM1 with single cysteine replacements at residues 246–249, as indicated, assessed as in Figure 2. The STIM1 dimer band is marked with an arrow. F, Single-cell [Ca²⁺]_i measurements in HeLa cells expressing wildtype GFP-STIM1 (WT; n = 55), GFP-STIM1(D247C) (D247C; n = 48), GFP-STIM1(L248C) (L248C; n = 50), and non-transfected HeLa cells (HeLa; n = 65). Cells were exposed to solutions containing varied concentrations of CaCl₂ or 1 µM thapsigargin (TG) as indicated. G,H, Confocal micrographs of HeLa cells expressing GFP-STIM1(L248C) (G) or GFP-STIM1(D247C) (H) at rest (−) and after store depletion (TG). Scale bars, 10 µm. I, Upper, Provisional model for helix-helix packing of the transmembrane helices and initial parts of CC1 in wildtype STIM1. Closely packed transmembrane helix residues (from Figure 2) are highlighted with grey boxes, predicted coiled-coil core residues in the juxtamembrane segment (from Figure S5) in red, and predicted CC1 core residues beyond L251 (from Figure S5) in grey. Grey and red highlights correspond to different heptad registers of the core residues. Hatched shading of S237 and L251 indicates that the spacing of these residues is compatible with either adjacent heptad assignment. Lower, An alternative model for helix-helix packing of the D247C mutant. Core residues are highlighted with grey boxes. The D>C replacement introduces a favorable core residue at position 247, and could permit a local adjustment in the region from residues 240–247 that creates a coiled coil lacking heptad discontinuities. See also Figures S2, S3, and S4.

Figure 5. Crosslinking of STIM1 juxtamembrane residues

A, Abbreviated alignment of STIM sequences from varied species. Further examples are shown in Figure S5. Residues are shown in black if COILS (input matrix MTIDK, using weights, window length 14) calculates a coiled-coil probability greater than 0.5. Other residues are shown in grey. Boxed residues are assigned to core a or d positions by COILS. The brown highlight indicates a break in the predicted heptad repeat that is conserved across these species. The species represented are Nematostella vectensis, starlet sea anemone; Lingula anatina, a lingulid brachiopod; Drosophila melanogaster, common fruit fly; Daphnia pulex, a water flea; Strigamia maritima, a centipede; Strongylocentrotus purpuratus, purple sea urchin; Homo sapiens, human. B, Crosslinking of STIM1 with single cysteine replacements at residues 237, 241, or 244. The STIM1 dimer band is marked with an arrow. Crosslinking at these specific positions was tested because residues 241 and 244 align with predicted coiled-coil core residues of STIM1 orthologues across a broad range of species (Figure S5), and the spacing of residue 237 from these predicted core positions is compatible with its placement in a core position. The residue corresponding to S237 is, in fact, predicted to be a core residue in a few species, but the conservation of a polar serine in most species and its proximity to the transmembrane segment render coiled-coil prediction uncertain. Data from three biological replicates. C, Upper, STIM1 sequence from residues 214–248. Closely packed transmembrane helix residues are highlighted with grey boxes, and juxtamembrane residues that crosslink are highlighted in red. There is a shift in the heptad repeat of helix-helix packing centered on positions 233 and 234. Hatched shading indicates that the spacing of the highlighted residue is compatible with either adjacent heptad assignment. Lower, Either residue 233 or residue 234 alone, as a core residue, could support continuation of an imperfect coiled coil across the region of ambiguity. See also Figures S5, S6, and S7.

Figure 6. Analysis of STIM1 residues Q233, N234, and S237

A, The effect of additional Q233L/S237L mutations on crosslinking of STIM1(A230C), assessed as in Figure 2. The STIM dimer band is marked with an arrow. Data representative of three biological replicates. B, Confocal micrographs of HeLa cells expressing GFP-STIM1(Q233L/S237L) under the conditions indicated. Scale bars, 10 µm. C, The effect of additional N234L/S237L mutations on crosslinking of STIM1(A230C), assessed as in Figure 2. The STIM dimer band is marked with an arrow. D, Confocal micrographs of HeLa cells expressing GFP-STIM1(N234L/S237L) under the conditions indicated. Scale bar, 10 µm. E, Single-cell [Ca²⁺]_i measurements in HeLa cells expressing wildtype GFP-STIM1 (WT; n = 55), GFP-STIM1(N234L/S237L) (N234L S237L; n = 73), and non-transfected HeLa cells (HeLa; n = 65). Cells were exposed to solutions containing varied concentrations of CaCl₂ or 1 µM thapsigargin (TG) as indicated. These experiments were performed together with those of Figure 4F, and the data plotted in Figure 4F for wildtype GFP-STIM1 and non-transfected HeLa cells are repeated here for reference. F, SDS-polyacrylamide gel analysis of the proteins used for energy transfer experiments. In the gel on the left, the samples in lanes 1–3 are the wildtype, L251S, and N234L/S237L proteins, respectively, stained with Coomassie Brilliant Blue. In the separate gel on the right, the sample in lane 4 is unlabeled wildtype protein as a negative control for fluorescence, and the samples in lanes 5–7 are the same fluorescein-labeled proteins as in lanes 1–3 of the Coomassie Brilliant Blue-stained gel. The gel was illuminated with UV light and the resulting fluorescence imaged to detect dye-labeled proteins. Inset: STIM1 proteins designed for Tb³⁺-acceptor energy transfer measurements comprise the STIM1 cytoplasmic domain, STIM1(233–685), with an engineered lanthanide-binding tag (LBT) at the N terminus to bind Tb³⁺ and a single engineered cysteine at the C terminus for covalent labelling with an acceptor dye. Fluorescein-5-maleimide was the labelling reagent in these experiments. G–I, Gated luminescence emission spectra of N234L/S237L (G), wildtype (H), and L251S (I) proteins labeled with donor Tb³⁺ and acceptor fluorescein. The spectrum of each protein labeled with donor Tb³⁺, but without acceptor, is shown for comparison (No FL). In (H), the specificity control with acceptor-labeled protein, but omitting Tb³⁺, is also shown. The arrows in G–I mark the expected position of the fluorescein emission peak. Data are representative of two biological replicates. See also Figure S6.

Figure 7. Summary diagram placing evidence from this study into context

The STIM1 transmembrane helix and the initial part of CC1, extending to the SOAR/CAD-interacting region, are shown. Residues found to be crosslinked in EGTA in this study are marked with arrowheads, demonstrating apposition of the transmembrane helices and initial parts of CC1 from residue 216 to 251. The crosslinking of 234C and 247C is discussed in the text. Assigned core positions (a or d) in the conserved coiled-coil organization of STIM1 orthologues (Figure 5A; Figure S5) are indicated. L251, L258, and L261 have been shown to be involved in CC1-SOAR/CAD interaction, implying that close apposition of the CC1 helices of active STIM1 might extend to residue 261. The highlighted shifts in the heptad repeat (beige boxes) may have evolved to facilitate switching between the inactive and active conformations of STIM1 and to facilitate the reversible binding and release of SOAR/CAD.