Interhelical interactions within the STIM1 CC1 domain modulate CRAC channel activation

Abstract

The calcium release activated calcium channel is activated by the endoplasmic reticulum-resident calcium sensor protein STIM1. On activation, STIM1 C terminus changes from an inactive, tight to an active, extended conformation. A coiled-coil clamp involving the CC1 and CC3 domains is essential in controlling STIM1 activation, with CC1 as the key entity. The nuclear magnetic resonance-derived solution structure of the CC1 domain represents a three-helix bundle stabilized by interhelical contacts, which are absent in the Stormorken disease-related STIM1 R304W mutant. Two interhelical sites between the CC1α₁ and CC1α₂ helices are key in controlling STIM1 activation, affecting the balance between tight and extended conformations. Nuclear magnetic resonance-directed mutations within these interhelical interactions restore the physiological, store-dependent activation behavior of the gain-of-function STIM1 R304W mutant. This study reveals the functional impact of interhelical interactions within the CC1 domain for modifying the CC1-CC3 clamp strength to control the activation of STIM1.

Extended Data Fig. 1. CC1 crystallographic structure and corresponding patch-clamp-data

a Top: Comparison of the published crystallographic structure fragment (PDB: 4O9B, blue) to the STIM1 CC1 NMR model. The positions of the residues mutated in the X-ray structure are labelled. The first five N-terminal residues (G229-F233) of the recombinant fragment, the remainder of the thrombin cleavage sequence, are not part of the native sequence. Bottom: Partial sequence alignment of the fragments used for NMR (top) and crystallography (bottom) with mutations indicated by red boxes. The molecular graphics were created by PyMOL (v. 2.3, Schrödinger, LLC). b Depiction of a patch clamp experiment using the whole cell configuration. The entry of Ca²⁺ ions through Orai1 channels generates an electrical current that is registered by an Ag/AgCl electrode. This electrode is inserted into a glass pipette that is sealed to the plasma membrane of a target cell. c Orai1 current activation shown by patch clamp recordings of N-terminally tagged CFP-Orai1 co-expressed with YFP-STIM1 M244L + L321M (pink). HEK293 cells were exclusively used for all recordings. The patch clamp experiment was replicated on two different days using independent transfections with the indicated number of cells (n). Data represent mean values ±SEM.

Extended Data Fig. 2. The solution NMR structure of STIM1 CC1

Extended Data Fig. 2 Side a and top b views of the solution structure of STIM1 CC1. Blue color represents residues that are in close NOE contact with the SDS detergent (the residues are listed in the table insert). The NMR experiments were recorded in presence of 7.0 mM SDS to avoid non-specific CC1 homo-oligomerization occurring in absence of the detergent. We note that at this concentration below the CMC (critical micelle concentration) of 8.2 mM (25 °C), SDS does not cause any secondary structure changes, as proven by CD spectra (Supplementary Fig. 1). The intermolecular NOEs observed between the surface-exposed residues of STIM1 CC1 and SDS molecules are consistent with protection of the highlighted (blue) residues listed in the table insert by the detergent, thus preventing CC1 homo-oligomerization while leaving intramolecular coiled-coil contacts intact.

Extended Data Fig. 3. Spectra of STIM1 CC1 wild-type and Stormorken mutant

Assigned 700 MHzH-¹⁵N HSQC spectra of 0.3 mM ¹⁵N-STIM1 CC1 wild-type a and Stormorken mutant b.

Extended Data Fig. 4. Secondary structure prediction of STIM1 CC1 wild-type and Stormorken mutant

Secondary structure prediction for STIM1 CC1 wild-type a and Stormorken mutant b from Talos-N. Green lines represent the order parameter S predicted from the chemical shifts. Red bars indicate the probability (in %*100) for residue to adapt a helical secondary structure.

Extended Data Fig. 5. STIM1 homomerization and Orai1 activation by STIM1 mutants

a STIM1 homomerization experiments of N-terminally tagged CFP- and YFP-STIM1 I290S + A293S ± R304W. Ca²⁺ store depletion was induced by perfusion with 1 μM thapsigargin in Ca²⁺ free solution. b Orai1 current activation shown by patch clamp recordings of N-terminally tagged CFP-Orai1 co-expressed with YFP-STIM1 L251S ± I290S + A293S. Color code: WT (black), L251S (purple), R304W (red), L251S + I290S + A293S (gray), I290S + A293S + R304W (blue), and I290S + A293S (magenta). HEK293 cells were exclusively used for all experiments. Experiments were replicated on at least two different days using independent transfections with the indicated number of cells (n). Data represent mean values ± SEM.

Extended Data Fig. 6. Graphical representation of NOE distance and hydrogen bond restraints

Graphical representation of NOE distance and hydrogen bond restraints used for the structure calculation. The parallel closely spaced lines indicate intra-helical (i to i+4) restraints from CS-Rosetta. The lines crossing each other near the center are characteristic of anti-parallel alignment of helices. The secondary structure ranges are indicated, as well as the non-native residues (G1-F5), by color coding corresponding to the Figures in the main text.

Fig. 1. STIM1 CC1 forms a compact three-helix bundle.

a Ribbon representation of 20 spatially aligned conformers of STIM1 CC1 fragment (backbone RMSD 1.5 Å for all residues, 0.51 Å for the α-helices, see Methods). b The lowest restraint violation energy structure (α₁: H240-E270, α₂: L282-L303 and α₃: A317-S340). The dashed line indicated the NOE contact between H272 and W341. The molecular graphics were created by PyMOL (v. 2.3, Schrödinger, LLC).

Fig. 2. Coiled-coil epitopes between α1 and α2 helices of CC1.

a, c Scheme of both coiled-coil regions identified in NMR; red lines denote the observed NOE contacts. b, d Top-views of these two coiled-coil motifs in the NMR solution model. e Side-view of the entire α₁-α₂ segment highlighting the coiled-coil interaction sites.

Fig. 3. Stormorken mutation alters CC contacts and increases loop₂ rigidity of CC1.

a CC1 secondary structure predicted from backbone chemical shifts The transparent helix (in violet) represents the predicted α-helical propensity in the Stormorken mutant. b Chemical shift differences (¹H, ¹⁵N) between STIM1 CC1 wild-type and Stormorken mutant. c Backbone relaxation dynamic profiles for wild-type (blue dots) and Stormorken mutant (red dots) of CC1. R₁/R₂ values were computed from 5 independent measurements each with error propagation (mean ± S.D.). d and e show carbon detected 2D CON 700 MHz NMR spectra of wild-type and Stormorken STIM1 CC1, respectively.

Fig. 4. CC contact mutations of CC1 reduce Orai1 activation.

a Mutation of the first (I290S + A293S) interhelical site. Left: Orai1 current activation shown by patch clamp recordings of N-terminally tagged CFP-Orai1 co-expressed with YFP-STIM1 I290S + A293S ± R304W. Right: Comparison of current activation levels at Time 0 vs. 190 seconds. b Mutation of the second (L300S + L303S) interhelical site. Left and Right: Same as in a but for CFP-Orai1 co-expressed with YFP-STIM1 L300S + L303S ± R304W. Color code: WT (black), R304W (red), I290S + A293S + R304W / L300S + L303S + R304W (blue / cyan), and I290S + A293S / L300S + L303S (magenta / orange). HEK293 cells were exclusively used for all experiments. Student’s two-tailed t-test was employed for statistical analyses with differences considered statistically significant at p<0.05. Asterisks (*) indicate significant difference. Exact p values are listed in Supplementary Table 4. Experiments were replicated on at least two different days using independent transfections with the indicated number of cells (n). Data represent mean values ± SEM.

Fig. 5. CC contact mutations restore OASF R304W tight state by reinforcing CC1-CC3 clamp.

a-c Mutation of the first (I290S + A293S) interhelical site. a and b Intramolecular FRET showing conformational changes of YFP-OASF-CFP I290S + A293S ± R304W in the absence (a) and presence (b) of unlabeled Orai1. c Intermolecular FIRE interaction indicating heteromerization of CFP-CC3 with YFP-CC1 I290S + A293S ± R304W. d-f Mutation of the second (L300S + L303S) interhelical site. Same as in a-c but for YFP-OASF-CFP L300S + L303S ± R304W (d) and (e) as well as CFP-CC3 with YFP-CC1 L300S + L303S ± R304W (f). For FIRE control experiments, a CFP construct lacking the CC3 domain was co-expressed with YFP-CC1. Color code: WT (black), R304W (red), I290S + A293S + R304W / L300S + L303S + R304W (blue / cyan), I290S + A293S / L300S + L303S (magenta / orange), and control (gray). HEK293 cells were exclusively used for all experiments. Student’s two-tailed t-test was employed for statistical analyses with differences considered statistically significant at p<0.05. Asterisks (*) indicate significant difference. Exact p values are listed in Supplementary Table 5. Experiments were replicated on at least two different days using independent transfections with the indicated number of cells (n). Data represent mean values ± SEM.

Fig. 6. Simplified model of STIM1 activation.

The CC1-CC3 clamp provides control over the release of CAD/SOAR from the CC1 domain. Interhelical interactions within the CC1 domain affect the strength of the CC1-CC3 clamp formation, the release of which leads to the exposure of the CAD/SOAR domain. The extended, activated state (right) can be switched to the tight, inactive states (left, middle) by weakening of CC1 interhelical interactions concomitantly strengthening CC1-CC3 interactions. Mutations stated at the bold arrows lead to the respective STIM1 activation states. For simplicity, the dimeric state of the CC1-CAD/SOAR fragments in the tight, store-dependent states is omitted, and the activated state might as well exhibit a crossing of the CC1 α₃.