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

Stathopulos PB, Schindl R, Fahrner M, Zheng L, Gasmi-Seabrook GM, Muik M, Romanin C, Ikura M - Nat Commun (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: University Health Network and Department of Medical Biophysics, Campbell Family Cancer Research Institute, Ontario Cancer Institute, University of Toronto, Room 4-804, MaRS TMDT, 101 College Street, Toronto, Ontario, Canada M5G 1L7.

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.

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NMR structures of apo CC1[TM-distal]-CC2 and the CC1[TM-distal]-CC2:Orai1 C272-292 complexa, Domain architecture of human STIM1. Amino terminus (N); signal peptide (S); canonical EF-hand (EF1); non-canonical EF-hand (EF2); sterile α motif (SAM); transmembrane segment (TM); putative coiled-coil (CC) 1, 2 and 3 (CC1; CC2; 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); transmembrane (TM) segments 1, 2, 3 and 4 (TM1, TM2, TM3, 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 Fig. S2c, S2d, S2e, S3 and S4). d, Supercoiling within the α1:α1’ interface (defg/abcdefg/a = 4/7/1). e, Supercoiling within α2:α2’ interface (abcdefg/abcd/abcd = 7/4/4). f, Cartoon view of the CC1[TMdistal]-CC2:Orai1 C272-292 structure. α1 Helix (α1); Loop 1 (L1); α2 helix (α2); Orai1 C272-292 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 C272-292 peptide which pack into the pocket are coloured salmon. h, Supercoiling within the α2:Orai1 C272-292 interface (defg/abcdefg/a = 4/7/1). In (d), (e) and (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.
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Figure 1: NMR structures of apo CC1[TM-distal]-CC2 and the CC1[TM-distal]-CC2:Orai1 C272-292 complexa, Domain architecture of human STIM1. Amino terminus (N); signal peptide (S); canonical EF-hand (EF1); non-canonical EF-hand (EF2); sterile α motif (SAM); transmembrane segment (TM); putative coiled-coil (CC) 1, 2 and 3 (CC1; CC2; 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); transmembrane (TM) segments 1, 2, 3 and 4 (TM1, TM2, TM3, 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 Fig. S2c, S2d, S2e, S3 and S4). d, Supercoiling within the α1:α1’ interface (defg/abcdefg/a = 4/7/1). e, Supercoiling within α2:α2’ interface (abcdefg/abcd/abcd = 7/4/4). f, Cartoon view of the CC1[TMdistal]-CC2:Orai1 C272-292 structure. α1 Helix (α1); Loop 1 (L1); α2 helix (α2); Orai1 C272-292 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 C272-292 peptide which pack into the pocket are coloured salmon. h, Supercoiling within the α2:Orai1 C272-292 interface (defg/abcdefg/a = 4/7/1). In (d), (e) and (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.

Mentions: STIM1 is a type I transmembrane protein with luminal EF-hand and sterile α-motif (SAM) domains (EF-SAM) that respond to Ca2+ depletion through intramolecular destabilization of the EF-hand:SAM interface, promoting intermolecular homomerization of this region in SOCE initiation 13,14. The cytosolic architecture of STIM1 is defined by three putative coiled-coil (CC) segments immediately following the single-pass transmembrane (TM) helix and a distal C-terminal Lys-rich region (poly-K) (Fig. 1a). STIM1 fragments lacking the luminal and TM domains can activate CRAC entry, independent of Ca2+ store depletion 15,16, implying the cytosolic portion is sufficient for eliciting SOCE. Specifically, the Orai activating STIM fragment (OASF) which includes CC1-CC2-CC3 (i.e. residues 233-450/474) encompasses the machinery required for Orai1 activation 17; further, the minimal boundaries within OASF required for coupling to and generation of Orai1 currents are found in the CC2-CC3 region. The STIM-Orai activating region (SOAR) 18, CRAC activating domain (CAD) 19 and coiled-coil boundary 9 (ccb9) 20 fragments defined by residues 344-442, 342-448 and 339-446, respectively, can maximally activate Orai1 currents in the absence of store depletion. A CC1-CC2-CC3 (i.e. residues 238-462) fragment only activates Orai1 currents after clustering; further, co-clustering of CC1 (i.e. residues 238-343) with a spontaneously Orai1-activating CC1-CC2-CC3 fragment (i.e. residues 315-462) inhibits Orai1 activity 21. Hence, while CC2-CC3 contains the minimum domains for Orai1 coupling and activation, the interplay between CC1, CC2 and CC3 modulates the quiescent and activation-competent states of CC2-CC3. Interestingly, a familial R429C mutation in CC3 linked with immunodeficiency and immune dysregulation has a dominant-negative effect on CRAC channel function in patients, without abrogation of full-length protein expression, suggesting a role in STIM1 multimerization and/or STIM1:Orai1 interactions for this CC3 residue position 22,23.


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

Stathopulos PB, Schindl R, Fahrner M, Zheng L, Gasmi-Seabrook GM, Muik M, Romanin C, Ikura M - Nat Commun (2013)

NMR structures of apo CC1[TM-distal]-CC2 and the CC1[TM-distal]-CC2:Orai1 C272-292 complexa, Domain architecture of human STIM1. Amino terminus (N); signal peptide (S); canonical EF-hand (EF1); non-canonical EF-hand (EF2); sterile α motif (SAM); transmembrane segment (TM); putative coiled-coil (CC) 1, 2 and 3 (CC1; CC2; 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); transmembrane (TM) segments 1, 2, 3 and 4 (TM1, TM2, TM3, 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 Fig. S2c, S2d, S2e, S3 and S4). d, Supercoiling within the α1:α1’ interface (defg/abcdefg/a = 4/7/1). e, Supercoiling within α2:α2’ interface (abcdefg/abcd/abcd = 7/4/4). f, Cartoon view of the CC1[TMdistal]-CC2:Orai1 C272-292 structure. α1 Helix (α1); Loop 1 (L1); α2 helix (α2); Orai1 C272-292 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 C272-292 peptide which pack into the pocket are coloured salmon. h, Supercoiling within the α2:Orai1 C272-292 interface (defg/abcdefg/a = 4/7/1). In (d), (e) and (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.
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Figure 1: NMR structures of apo CC1[TM-distal]-CC2 and the CC1[TM-distal]-CC2:Orai1 C272-292 complexa, Domain architecture of human STIM1. Amino terminus (N); signal peptide (S); canonical EF-hand (EF1); non-canonical EF-hand (EF2); sterile α motif (SAM); transmembrane segment (TM); putative coiled-coil (CC) 1, 2 and 3 (CC1; CC2; 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); transmembrane (TM) segments 1, 2, 3 and 4 (TM1, TM2, TM3, 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 Fig. S2c, S2d, S2e, S3 and S4). d, Supercoiling within the α1:α1’ interface (defg/abcdefg/a = 4/7/1). e, Supercoiling within α2:α2’ interface (abcdefg/abcd/abcd = 7/4/4). f, Cartoon view of the CC1[TMdistal]-CC2:Orai1 C272-292 structure. α1 Helix (α1); Loop 1 (L1); α2 helix (α2); Orai1 C272-292 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 C272-292 peptide which pack into the pocket are coloured salmon. h, Supercoiling within the α2:Orai1 C272-292 interface (defg/abcdefg/a = 4/7/1). In (d), (e) and (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.
Mentions: STIM1 is a type I transmembrane protein with luminal EF-hand and sterile α-motif (SAM) domains (EF-SAM) that respond to Ca2+ depletion through intramolecular destabilization of the EF-hand:SAM interface, promoting intermolecular homomerization of this region in SOCE initiation 13,14. The cytosolic architecture of STIM1 is defined by three putative coiled-coil (CC) segments immediately following the single-pass transmembrane (TM) helix and a distal C-terminal Lys-rich region (poly-K) (Fig. 1a). STIM1 fragments lacking the luminal and TM domains can activate CRAC entry, independent of Ca2+ store depletion 15,16, implying the cytosolic portion is sufficient for eliciting SOCE. Specifically, the Orai activating STIM fragment (OASF) which includes CC1-CC2-CC3 (i.e. residues 233-450/474) encompasses the machinery required for Orai1 activation 17; further, the minimal boundaries within OASF required for coupling to and generation of Orai1 currents are found in the CC2-CC3 region. The STIM-Orai activating region (SOAR) 18, CRAC activating domain (CAD) 19 and coiled-coil boundary 9 (ccb9) 20 fragments defined by residues 344-442, 342-448 and 339-446, respectively, can maximally activate Orai1 currents in the absence of store depletion. A CC1-CC2-CC3 (i.e. residues 238-462) fragment only activates Orai1 currents after clustering; further, co-clustering of CC1 (i.e. residues 238-343) with a spontaneously Orai1-activating CC1-CC2-CC3 fragment (i.e. residues 315-462) inhibits Orai1 activity 21. Hence, while CC2-CC3 contains the minimum domains for Orai1 coupling and activation, the interplay between CC1, CC2 and CC3 modulates the quiescent and activation-competent states of CC2-CC3. Interestingly, a familial R429C mutation in CC3 linked with immunodeficiency and immune dysregulation has a dominant-negative effect on CRAC channel function in patients, without abrogation of full-length protein expression, suggesting a role in STIM1 multimerization and/or STIM1:Orai1 interactions for this CC3 residue position 22,23.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: University Health Network and Department of Medical Biophysics, Campbell Family Cancer Research Institute, Ontario Cancer Institute, University of Toronto, Room 4-804, MaRS TMDT, 101 College Street, Toronto, Ontario, Canada M5G 1L7.

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.

Show MeSH
Related in: MedlinePlus