Initial activation of STIM1, the regulator of store-operated calcium entry

Abstract

Physiological Ca(2+) signaling in T lymphocytes and other cells depends on the STIM-ORAI pathway of store-operated Ca(2+) entry. STIM1 and STIM2 are Ca(2+) sensors in the endoplasmic reticulum (ER) membrane, with ER-luminal domains that monitor cellular Ca(2+) stores and cytoplasmic domains that gate ORAI channels in the plasma membrane. The STIM ER-luminal domain dimerizes or oligomerizes upon dissociation of Ca(2+), but the mechanism transmitting activation to the STIM cytoplasmic domain was previously undefined. Using Tb(3+)-acceptor energy transfer, we show that dimerization of STIM1 ER-luminal domains causes an extensive conformational change in mouse STIM1 cytoplasmic domains. The conformational change, triggered by apposition of the predicted coiled-coil 1 (CC1) regions, releases the ORAI-activating domains from their interaction with the CC1 regions and allows physical extension of the STIM1 cytoplasmic domain across the gap between ER and plasma membrane and communication with ORAI channels.

Figure 1. STIM1-PIP2 interaction

(A) GFP-STIM1^CT binding to PIP2 and other lipids arrayed on a lipid strip. GFP-STIM1^CT lacking the C-terminal polybasic segment (GFP-STIM1^CT-ΔK) and GFP alone are controls. (B) GFP-STIM1^CT binding to nanodiscs. Left, schematic of the experiment. A fluorescent phospholipid analog (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein)) serves as donor, and tetramethylrhodamine (TMR) attached to STIM1^CT as acceptor. Center and right, binding measurements on control and PIP2-containing nanodiscs. Recorded spectra are for nanodiscs alone (green), nanodiscs together with STIM1^CT (red), and STIM1^CT alone (black). In the subtracted spectra (lavender), donor fluorescence has been removed from the nanodisc + STIM1^CT spectra by subtracting an appropriately scaled nanodisc-alone spectrum. Only nanodiscs containing PIP2 exhibit energy transfer (right panel, compare lavender curve with black curve) confirming the specific association of GFP-STIM1^CT with PIP2 in bilayers. (C) GFP-STIM1^CT binding to liposomes. Upper, schematic of the equilibrium dialysis experiment. One chamber was loaded with PIP2-containing liposomes, the other chamber with control liposomes. The initial concentration of STIM1^CT in the two chambers was identical. Lower, the excess of GFP-STIM1^CT recovered from the PIP2 chamber after equilibration, as a fraction of total GFP-STIM1^CT in both chambers. (D) Exposure of the STIM1^CT C terminus assessed with an environment-sensitive probe. Upper, fluorescence spectra of IAEDANS and of AEDANS covalently attached to the indicated STIM1^CT proteins at introduced residue cysteine-686. Lower, wavelengths of peak fluorescence emission. The shift of the peak to a shorter wavelength in the wildtype protein reports partial burial of the fluorophore. Representative of two experiments.

Figure 2. Distance measurements in STIM1 cytoplasmic domain

(A) Schematic of STIM1^CT donor–acceptor labelling. STIM1^CT(C437S) was engineered with a lanthanide-binding tag (LBT) at its N terminus for labelling with donor Tb³⁺ and an added cysteine residue at its C terminus for labelling with acceptor fluorophore [see also Supplementary Figure 6]. The LBT is an engineered EF-hand with high affinity for Tb³⁺ and with a tryptophan residue positioned to serve as an antenna for excitation of Tb³⁺. The sequence of murine STIM1^CT used in these experiments is closely similar to human STIM1^CT throughout, and in particular is identical in CC1 and differs by a single K371R substitution in SOAR(CAD). CC1, predicted coiled coil region 1; SOAR, STIM1 Orai activating region; K, C-terminal polybasic tail. (B) Size-exclusion chromatography of wildtype and L251S variant STIM1^CT proteins. (C) Thermal melting monitored as change in circular dichroism at 222 nm. High-resolution thermal melting measurements detect a difference in stability between wildtype STIM1^CT and the activated variant L251S at temperatures below 40°C. (D) Gated luminescence spectra of labelled STIM1^CT proteins. The spectra were collected after 200 µs to eliminate light scattering and directly excited acceptor fluorescence. BODIPY-FL acceptor emission from the labelled wildtype protein is indicated (green arrow). (E) Luminescence decay of the indicated STIM1^CT proteins, followed at the donor wavelength in the absence and presence of acceptor (τ_D, τ_DA) and at the acceptor wavelength (τ_AD). Acceptor decays correspond well to donor decays, except that the 0.21-ms component accounts for a larger fraction of the total amplitude. This difference is expected. The physical basis for the exaggeration of the rapid component in acceptor decay traces has been detailed in. Residuals indicate no systematic deviation of the data from the fitted curves. (F) Cartoon interpreting the results of panels 2D and 2E. The distance measured between residues 233 and 686 implies that the wildtype protein is folded back, whereas the “activated” L251S protein is extended.

Figure 3. Lack of detectable CC1–CC1 association

(A) Recombinant CC1 is monomeric. SEC-MALS determination of CC1 molecular mass (right axis) is plotted with the UV absorbance trace indicating the protein peak (left axis). Inset, SDS-polyacrylamide gel analysis of the purified CC1 protein. (B) Schematic of STIM1^CT heterodimer in which one monomer was tagged at its N terminus with an LBT and the other monomer with a HAP peptide that binds the 8 kDa ligand α-bungarotoxin (BTX) with high affinity. The introduced cysteine residues used to crosslink the N termini are not shown. (C) SDS-polyacrylamide gel analysis, documenting that the heterodimer, as prepared, has roughly equal amounts of LBT-STIM1^CT and HAP-STIM1^CT. (D) Size-exclusion chromatography shows that bound fluorescent α-bungarotoxin forms a stable complex with STIM1^CT protein. The UV absorbance signal from protein (black curve and left axis) and the fluorescence signal from Alexa Fluor 488-labelled α-bungarotoxin in fractions eluting from the column (green symbols and right axis) are plotted against elution volume. (E) CC1 regions are not associated in dimeric STIM1^CTLeft, gated fluorescence spectra of the heterodimer with Tb³⁺ donor alone (black), uncrosslinked heterodimer with Tb³⁺ donor and fluorescent α-bungarotoxin acceptor (green), and N-terminally crosslinked heterodimer with Tb³⁺ donor and fluorescent α-bungarotoxin acceptor (red). Right, corresponding luminescence decay curves. Tb³⁺ donor emission was monitored except in the case labelled τ_AD, for which acceptor emission was monitored. Energy transfer between Tb³⁺ and fluorescent α-bungarotoxin was observed when the N termini of the heterodimer were artificially apposed by forming a disulfide link, verifying that the assay detects CC1–CC1 proximity. However, there was no intradimer energy transfer in the absence of crosslinking. (F) Cartoon illustrating the conclusion that the N termini of the individual STIM1^CT monomers are not in close proximity in the STIM1^CT dimer.

Figure 4. Effects of forced CC1–CC1 association

(A) Nonreducing SDS-polyacrylamide gel verifying efficient disulfide crosslinking of CC1. (B) Far-UV CD spectra of uncrosslinked and crosslinked CC1. The dimer shows a modest increase in α-helix content, evident in the change in molar ellipticity at 208 nm and 222 nm. (C) Thermal melting of CC1 and crosslinked CC1 monitored at 222 nm. The dimer shows pronounced stabilization of a part of its α-helical structure. (D) Nonreducing SDS-polyacrylamide gel verifying efficient disulfide crosslinking of CC1(L251S). (E) Far-UV CD spectra of uncrosslinked and crosslinked CC1(L251S). The disulfide-linked CC1(L251S) dimer shows little change in α-helix content. (F) Thermal melting of CC1(L251S) and crosslinked CC1(L251S) monitored at 222 nm. In contrast to the finding with wildtype CC1 (panel 3D), disulfide crosslinking fails to stabilize the secondary structure of CC1(L251S). (G) Cartoon of CC1 as the monomer or as the disulfide-linked dimer. A straightforward interpretation of the differing results in panels 3D and 3G is that the increase in α-helix content and the stabilization of secondary structure occurs as a result of coiled coil formation adjacent to the site of crosslinking, (H) Binding of CC1, CC1(L251S), and crosslinked dimeric CC1 to immobilized MBP-SOAR. Left, the indicated samples were analyzed on a nonreducing SDS-polyacrylamide gel. Representative of three experiments for CC1 and dimeric CGG-CC1, and two experiments for CC1(L251S). Right, quantitation of the fraction of CC1 bound to MBP-SOAR.

Figure 5. Intradimer CC1–CC1 association triggers extension of the STIM1 cytoplasmic domain

(A) Schematic illustrating the placement of labels, with an LBT binding Tb³⁺ at the N terminus of STIM1^CT and a HAP tag binding Alexa Fluor 488-labelled α-bungarotoxin at the C terminus. To allow crosslinking in this experiment, a cysteine residue was introduced at the extreme N terminus of STIM1^CT. (B) Reducing SDS-polyacrylamide gel documenting the effectiveness of crosslinking after a 16-h reaction. (C) CC1–CC1 crosslinking abolishes energy transfer between N-terminal donor and C-terminal acceptor. Left, gated fluorescence spectra of the iodoacetamide-blocked (red) and crosslinked (black) samples. Right, corresponding luminescence decay curves. Tb³⁺ donor emission was monitored except in the trace labelled τ_AD (green), for which acceptor emission was monitored. (D) Cartoon interpreting the results of panel 5C. CC1–CC1 crosslinking, like the L251S mutation, leads to an extended conformation of STIM1^CT.

Figure 6. Model for STIM1 activation in cells

Left, inactive STIM1, with the individual CC1 regions (red and yellow) interacting with the SOAR(CAD) domains (magenta) (Figure 4H), a relatively short distance between the N terminus of CC1 and the STIM1 C-terminal polybasic tail (blue) (Figure 2D–E), and the polybasic tail partially buried (Figure 1D). STIM1 ER-luminal domain (brown) and SOAR(CAD) structures are as reported in the literature^16,53. Detailed structural information for CC1 and for the region (black) C-terminal to SOAR(CAD) is not available. The model is not intended to specify the configuration of the polypeptide backbone that links CC1 to SOAR(CAD), or the surface(s) of SOAR(CAD) that are in contact with CC1. Right, an alternative possibility for inactive STIM1, with the N termini of its two CC1 segments separated (Figure 3E). Note that if L251 and SOAR(CAD) interact directly (Figure 4H), the geometry of SOAR(CAD) in this case is likely to differ from that of the crystallized domain depicted. Center, active STIM1, with the initial portions of CC1 (red) coming together in a coiled coil (Figure 4A–F), a loss of the CC1–SOAR(CAD) interaction (Figure 4H), and an increased distance between the N terminus of CC1 and the polybasic tail (Figure 5C). The structures of the luminal domain and of SOAR(CAD), rendered as solid dimers in active STIM1, have not been determined.