STIM1/Orai1 coiled-coil interplay in the regulation of store-operated calcium entry

Abstract

Orai1 calcium channels in the plasma membrane are activated by stromal interaction molecule-1 (STIM1), an endoplasmic reticulum calcium sensor, to mediate store-operated calcium entry (SOCE). The cytosolic region of STIM1 contains a long putative coiled-coil (CC)1 segment and shorter CC2 and CC3 domains. Here we present solution nuclear magnetic resonance structures of a trypsin-resistant CC1-CC2 fragment in the apo and Orai1-bound states. Each CC1-CC2 subunit forms a U-shaped structure that homodimerizes through antiparallel interactions between equivalent α-helices. The CC2:CC2' helix pair clamps two identical acidic Orai1 C-terminal helices at opposite ends of a hydrophobic/basic STIM-Orai association pocket. STIM1 mutants disrupting CC1:CC1' interactions attenuate, while variants promoting CC1 stability spontaneously activate Orai1 currents. CC2 mutations cause remarkable variability in Orai1 activation because of a dual function in binding Orai1 and autoinhibiting STIM1 oligomerization via interactions with CC3. We conclude that SOCE is activated through dynamic interplay between STIM1 and Orai1 helices.

Figure 1. NMR structures of apo CC1_[TM-distal]-CC2 and the CC1_[TM-distal]-CC2:Orai1 C_272–292 complex.

(a) Domain architecture of human STIM1. Amino terminus (N); signal peptide (S); canonical EF-hand (EF1); non-canonical EF-hand (EF2); SAM; TM; putative CC1, CC2 and CC3, respectively; Pro/Ser-rich region; Lys-rich region (poly-K); carboxy terminus (C). Residue ranges are indicated above the domain diagram. Constructs employed in this study are shown below (cyan rectangles) with the residue range (black font) and nomenclature (cyan font) indicated. (b) Domain architecture of human Orai1. Amino terminus (N); TM segments 1, 2, 3 and 4 (TM1, TM2, TM3 and TM4, respectively); carboxy terminus (C). Residue ranges are indicated above the domain diagram. The yellow box delineates the fragment used in this study. (c) Cartoon view of the CC1_[TM-distal]-CC2 structure. α1 Helix (α1); loop 1 (L1); α2 helix (α2). Comprehensive structural validation was performed (Supplementary Figs S2c–e, S3 and S4). (d) Supercoiling within the α1:α1′ interface (defg/abcdefg/a=4/7/1). (e) Supercoiling within the α2:α2′ interface (abcdefg/abcd/abcd=7/4/4). (f) Cartoon view of the CC1_[TM-distal]-CC2:Orai1 C_272–292 structure. α1 Helix (α1); Loop 1 (L1); α2 helix (α2); Orai1 C_272–292 helix (O1). (g) Zoomed view of the SOAP shown in f (broken black boxes). The N-terminal α2 and C-terminal α2′ side chains (sticks) forming one Orai1-binding site are coloured teal. The side chains (sticks) of the Orai1 C_272–292 peptide, which pack into the pocket, are coloured salmon. (h) Supercoiling within the α2:Orai1 C_272–292 interface (defg/abcdefg/a=4/7/1). In d,e,h, the helical wheels show the heptad positions with only reciprocating ‘a’ (purple) and ‘d’ (magenta) packing residues adjacent to one another, not all four residues making up the hole; see Supplementary Fig. S5a, S5b and S5d for the proximity and orientation of the ‘a’ and ‘d’ side chains.

Figure 2. CC1_[TM-distal]-CC2 structural changes associated with Orai1 C_272–292 binding.

(a) V324 and L328′ side-chain (green sticks) proximity in the apo α1:α1′ interface. The distance between the V324-C_β and L328′-C_γ atoms is indicated (broken black line). (b) V324 and L328′ side-chain (green sticks) proximity in the α1:α1′ interface of the CC1_[TM-distal]-CC2:Orai1 C_272–292 structure. The distance between the V324-C_β and L328′-C_γ atoms is indicated (red broken line). (c) Central pivot point of the apo α2:α2′ interface. The intermolecular Y362-OH (green sticks) distance (broken black line) is shown. (d) Central α2:α2′ pivot point in the CC1_[TM-distal]-CC2:Orai1 C_272–292 structure. The intermolecular Y362-OH (green sticks) distance (broken red line) is shown. The Orai1 L273-C_γ (brown sticks) to Y362-OH and intermolecular Y361-OH (green sticks) distances (broken black lines) are also shown. (e) Distance between the α2 helical axes in the apo (blue cartoons; broken black line) versus Orai1 C_272–292-bound (white cartoons; broken red line) states. (f) Angular opening (broken curved line) of the C-terminal α2 region upon Orai1 C_272–292 binding. The apo α2 helices (blue cartoons) are shown relative to Orai1-bound α2 helices (white cartoons). (g) Surface electrostatics of the CC1_[TM-distal]-CC2 structure. The distinct α1:α1′ acidic and the C-terminal basic residues are labelled. (h) Electrostatic complementarity between CC1_[TM-distal]-CC2 and Orai1 C_272–292 derived from the complex structure. The basic rim residues of the SOAP (broken black circle) and acidic patches are labelled. The Orai1 C_272–292 peptides (yellow cartoons) and the acidic side chains are shown (red space fill). The electrostatic gradient in g,h is from −2 (red) to +2 (blue) kT/e.

Figure 3. Affects of CC1_[TM-distal]-CC2:Orai1 C_272–292 mutations on full-length function.

(a) Whole HEK-293 cell patch-clamp configuration used in this study. Current densities were measured at −86 mV. (b–f) Inward-current plots of cells co-overexpressing wild-type YFP-Orai1 and full-length mCh-STIM1 4EQ (b), V324P (c), Y361K/Y362K, Y362K or Y361K (d), A380R, I383R or 4KE (e) and L347R or L351R (f). (g,h) Inward-current plots of cells co-overexpressing wild-type mCh-STIM1 and mutant YFP-Orai1 R281A, L286S or R289A (g) and D284A/D287A/D291A or E272A/E275A/E278A (h). (i) Summary graph of maximal inward currents. Green bars represent spontaneous maximally activated currents and red bars indicate significantly attenuated maximal currents. The Orange bar represents I383R, which showed spontaneous inward currents slightly above background. Data are means±s.e.m. for n, number of cells and asterisks denote *P<0.05 by two-tailed Student’s t-test. Curve colours match the residues in Supplementary Fig. S9a,b. See Supplementary Table S3 for in vitro (Supplementary Figs S10 and S11) and live-cell data summary.

Figure 4. Docking of CC1_[TM-distal]-CC2 on the Orai hexamer.

(a) Structural homology between D. melanogaster Orai and human Orai1 C_272–292. The D. melanogaster Orai dimer (top) shows an antiparallel C-terminal configuration (yellow) highly homologous to human Orai1 C_272–292 (bottom; yellow). The analogous interhelix angles indicated (broken curved lines). (b) Docking of CC1_[TM-distal]-CC2 onto hexameric D. melanogaster Orai. The Orai1 C_272–292 helices within the CC1_[TM-distal]-CC2 complex were structurally aligned through sequentially similar regions in each D. melanogaster Orai dimer. CC3 locations are inferred from the position of the α2 C-termini. The Orai dimer unit is indicated (broken black box). (c) Sequence alignment of D. melanogaster Orai and H. sapiens Orai1 C-terminal residues. The H. sapiens Orai1 C_272–292 residues and the homologous residues visible in the D. melanogaster crystal structure are yellow. The boxed residues indicate the structurally aligned regions.

Figure 5. Schematic of STIM1/Orai1 CC interplay in SOCE regulation. CC interactions in the assembly of dimers (a) and hexamers (b).

The SOAP is indicated (broken black circle). STIM1 domains from individual subunits are cyan and light blue, while the Orai1 C-terminal domains are yellow. Sites of CC interactions are indicated (solid black lines). The assembly of STIM1 dimer units occurs through CC3:CC3′ interactions (broken black lines). Yellow cylinders represent the Orai1 channel with each dimer unit separated by a broken line. Channels are closed in the absence of STIM1 binding; however, interactions through CC1_[TM-distal]-CC2:Orai1 C_272–292 supercoiling (elucidated herein) and via Orai1 N-terminal domain (currently unresolved) open the channel pore. CC1 refers to the CC1_[TM-distal] region; CC1_{[TM-proximal]} interactions known to have a vital role in the quiescent-to-active conformational switch are not depicted.