A coiled-coil clamp controls both conformation and clustering of stromal interaction molecule 1 (STIM1)

Abstract

Store-operated Ca(2+) entry, essential for the adaptive immunity, is initiated by the endoplasmic reticulum (ER) Ca(2+) sensor STIM1. Ca(2+) entry occurs through the plasma membrane resident Ca(2+) channel Orai1 that directly interacts with the C-terminal STIM1 domain, named SOAR/CAD. Depletion of the ER Ca(2+) store controls this STIM1/Orai1 interaction via transition to an extended STIM1 C-terminal conformation, exposure of the SOAR/CAD domain, and STIM1/Orai1 co-clustering. Here we developed a novel approach termed FRET-derived Interaction in a Restricted Environment (FIRE) in an attempt to dissect the interplay of coiled-coil (CC) interactions in controlling STIM1 quiescent as well as active conformation and cluster formation. We present evidence of a sequential activation mechanism in the STIM1 cytosolic domains where the interaction between CC1 and CC3 segment regulates both SOAR/CAD exposure and CC3-mediated higher-order oligomerization as well as cluster formation. These dual levels of STIM1 auto-inhibition provide efficient control over the coupling to and activation of Orai1 channels.

Keywords: Calcium Release-activated Calcium Channel Protein 1 (ORAI1); Fluorescence; Patch Clamp; Signal Transduction; Stromal Interaction Molecule 1 (STIM1).

FIGURE 1.

Coiled-coil 1 controls formation of CAD clusters. A, scheme of STIM1 depicting individual domains: ER luminal EF hand and SAM domain, transmembrane domain (TM), coiled-coil (CC) domains: CC1 subdivided into α1, α2, α3, respectively, CC2, CC3, Pro/Ser-rich domain, Lys-rich domain. Numbers correspond to amino acid positions of human STIM1. Below, solution NMR structures of STIM1 N-terminal domains as well as of the CC1_α3-CC2 fragment, and the crystal structure of SOAR are magnified. B–E, FIRE system: schemes of FIRE constructs (Y-TMG-x; x: OASF, CAD, CC3₄₄₉) used for fluorescence imaging experiments. Y (YFP) is located in the ER lumen, STIM1 fragments are located on the cytosolic side, attached via a Gly-linker to the ER transmembrane segment. All constructs were overexpressed in HEK 293 cells, and representative images are depicted: (B) Y-TMG (control) showed ER distribution without cluster formation. (C) Y-TMG-OASF resulted in ER distribution and no cluster formation. Y-TMG-CAD (D) and Y-TMG-CC3₄₄₉ (E) revealed clear cluster formation. The bar in each fluorescence image corresponds to 5 μm. F, far-UV-CD spectrum of STIM1 CC3ext. The negative ellipticity minima at 208 and 222 nm are indicative of an α-helical fold. The spectrum was acquired at 0.25 mg ml⁻¹ and is an average of 3 scans. G, thermal stability of STIM1 CC3ext. The sigmoidal unfolding profile suggests a cooperative unfolding and is consistent with the high α-helicity of CC3ext. The thermal melt was acquired at 0.25 mg ml⁻¹. H, SEC of STIM1 CC3ext. After 100-μl injections of 1.25 mg ml⁻¹ (black trace) and 0.25 mg ml⁻¹ (red trace), CC3ext eluted in the void volume of the Superdex 200 10/30 GL column (blue vertical broken line) indicative of higher order oligomers with a molecular mass > 600 kDa. The expected elution volume of monomeric CC3ext is shown for reference (green vertical broken line). Inset depicts a Coomassie Blue-stained SDS-Page gel for fractions recovered following gel filtration showing monomeric CC3ext of 12.2 kDa.

FIGURE 2.

ER-anchored CAD or OASF co-localizes with and constitutively activates Orai1 channels. A, time course of inward currents from HEK293 cells co-expressing fluorescently labeled TMG-CAD + Orai1 and TMG-OASF + Orai1, as well as STIM1 or Orai1 as control, and (B) the respective current-voltage relationship. Fluorescence images showing localization and overlay of (C) C-Orai1 + Y-TMG-OASF, (D) C-Orai1 + Y-TMG-CAD, and (E) C-Orai1 + Y-TMG-CC3₄₄₉. The bar in each fluorescence image corresponds to 5 μm.

FIGURE 3.

Deletion of CC1_α1 leads to STIM1 C-terminal extension and results in constitutive coupling to and full activation of Orai1 channels. Time course of whole-cell inward currents from HEK293 cells co-expressing Orai1 together with (A) wild-type STIM1, STIM1 ΔCC1_α1, and STIM1 ΔCC1_α1α2 as well as (B) STIM1 ΔCC1_α2, STIM1 ΔCC1_α3 or STIM1 ΔCC1_α2α3. The respective life cell image series (C–E) show localization and overlay under resting cell conditions (upper panel) and following 5 min store depletion with 2 μm TG in nominally free extracellular Ca²⁺ solutions (lower panel). F, block diagram summarizing FRET values (E_app) of double-labeled OASF wild-type and deletion mutants (numbers in brackets indicate the quantity of cells measured): Y-OASF-C (WT), Y-OASF ΔCC1_α1-C, Y-OASF ΔCC1_α2-C, Y-OASF ΔCC1_α3-C, Y-OASF ΔCC1_α1α2-C, and Y-OASF ΔCC1_α2α3-C. G–I, representative localization and calculated FRET image are shown for Y-OASF-C (WT), Y-OASF ΔCC1_α1-C, and Y-OASF ΔCC1_α2α3-C. Calibration bar is 5 μm throughout.

FIGURE 4.

Homomerization potential of coiled-coil domains as well as helical segments and their heteromerization with CC1_α1. A, FIRE system: scheme of FIRE constructs illustrating background FRET (control, left) and FRET derived from a specific interaction (right). B, scheme of human STIM1 OASF depicting CC1, CC2 and CC3 domains. The same color coding for respective domains is used in C and D. C, homomerization potential of individual CC or helical fragments (CC1_α1 aa 233–276; CC1_α2 aa 273–309, CC1_α3 aa 303–342) determined by FIRE. D, block diagram depicting heteromeric interactions, obtained by FIRE, of CC1_α1 with: TMG (control), CC1_α2, CC1_α3, CC2, CC3₄₂₀, CC3₄₃₀, and CC3₄₄₉. Dashed lines in C and D represent the magnitude of the background signal. Number of cells studied are given in brackets. E–G, representative fluorescence images of Y-TMG-CC3 constructs of various lengths: (E) Y-TMG-CC3₄₂₀ (no cluster formation), (F) Y-TMG-CC3₄₃₀ (partial cluster formation), and (G) Y-TMG-CC3₄₄₉ (strong cluster formation). H, representative fluorescence images of co-expressed Y-TMG-CC1_α1 + Y-TMG-CC3₄₃₀.revealing ER distribution without cluster formation. Calibration bar is 5 μm throughout.

FIGURE 5.

Heteromeric interactions of the respective α₂ and α₃ helix of the CC1 domain with various OASF domains. FRET (E_app) determined from heteromeric interactions of (A) CC1 _α2 and (B)CC1 _α3 with various domains of OASF as depicted in Fig. 4. Interaction with TMG (no OASF fragment linked) represents control, with the dashed lines in A and B representing the magnitude of the background signal. Number of cells studied are given in brackets.

FIGURE 6.

The CC1_α1 domain is sufficient for controlling STIM1 conformational transition from a quiescent to an activated state. A, schematic diagram of human STIM1 OASF with highlighted point mutations that either weaken (red L251S) or enhance (green R426L) coiled-coil stability. B, block diagram summarizing FRET values (E_app) of double-labeled Y-OASF ΔCC1_α2α3-C (WT) as well as inserted point mutations (single L251S, single R426L, double L251S R426L). C, time courses of inward currents from whole-cell patch-clamp experiments co-expressing Orai1 with full-length STIM1 or STIM1 ΔCC1_α2α3 or with the point mutation L251S introduced into STIM1 ΔCC1_α2α3 that resulted in constitutively active inward currents. D, R426L mutation introduced either in wild type STIM1 or STIM1 ΔCC1_α2α3 yielded highly attenuated inward Ca²⁺ currents when co-expressed with Orai1, while the additional point mutation L251S restored inward currents to similar levels in full length as well as STIM1 ΔCC1_α2α3 L251S R426L mutants.

FIGURE 7.

Release of CC1_α1-CC3 interaction is required for subsequent CC3₄₄₉-mediated cluster formation. Block diagrams summarizing the homomerization (A) as well as heteromerization potentials (B) of various CC or helical STIM1 fragments with individual point mutations as determined by FIRE. The dashed line in A represents the magnitude of the background signal. Fluorescence images from cells expressing (A, lower panel) Y-TMG-CC3₄₃₀ R426L revealed cluster formation whereas cells co-expressing (B, lower panel) C-TMG-CC1_α1 + Y-TMG-CC3₄₃₀ R426L led to ER localization without cluster formation. C, representative fluorescence images from cell expressing Y-TMG-OASF L251S exhibited clear cluster formation while (D) Y-TMG-OASF R426L showed ER localization without clusters. Calibration bar is 5 μm throughout.

FIGURE 8.

Scheme depicting human STIM1 activation mechanism. A, scheme of STIM1 depicting individual domains as well as positions L251 and R426, as denoted in Fig. 1A. B–D, hypothetical model reconciling the available key high resolution structural data for depicting structural changes and activation of human STIM1, following ER Ca²⁺ store depletion (left to right). B, in resting cells, the inactive quiescent form of STIM1 (STIM1 Tight State) is mainly accomplished via a coiled-coil clamp by heteromeric interaction between CC1_α1 (yellow) and CC3 (red) resulting in a tight conformation. Residues at positions 251 and 426 are suggested as molecular determinants of the CC1_α1-CC3 interface. We illustrated the V-shaped CAD/SOAR structure in a top-down position (Λ-shaped) facing the ER membrane with its positive charges. C, upon store depletion, a conformational change (STIM1 Extended State) is accomplished due to a release of the heteromeric CC1_α1-CC3 clamp concomitant to a homomeric CC1_α1-CC1_α1 assembly, initiated by homomerization of the ER luminal part. D, finally, the STIM1 Oligomerized State is achieved via CC3 homomerization connecting STIM1 dimers in the oligomerized structure for coupling to and activation of Orai1 channels. For simplicity, STIM1 oligomerization is only depicted for two Orai1 dimers, with a suggested assembly of three Orai1 dimers with six STIM1 in a hexametric channel complex .