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STIM1 couples to ORAI1 via an intramolecular transition into an extended conformation.

Muik M, Fahrner M, Schindl R, Stathopulos P, Frischauf I, Derler I, Plenk P, Lackner B, Groschner K, Ikura M, Romanin C - EMBO J. (2011)

Bottom Line: The C-terminal rearrangement of STIM1 does not require a functional CRAC channel, suggesting interaction with ORAI1 as sufficient for this conformational switch.Corresponding full-length STIM1 mutants exhibited enhanced interaction with ORAI1 inducing constitutive CRAC currents, even in the absence of store depletion.We suggest that these mutant STIM1 proteins imitate a physiological activated state, which mimics the intramolecular transition that occurs in native STIM1 upon store depletion.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biophysics, University of Linz, Linz, Austria.

ABSTRACT
Stromal interaction molecule (STIM1) and ORAI1 are key components of the Ca(2+) release-activated Ca(2+) (CRAC) current having an important role in T-cell activation and mast cell degranulation. CRAC channel activation occurs via physical interaction of ORAI1 with STIM1 when endoplasmic reticulum Ca(2+) stores are depleted. Here we show, utilizing a novel STIM1-derived Förster resonance energy transfer sensor, that the ORAI1 activating small fragment (OASF) undergoes a C-terminal, intramolecular transition into an extended conformation when activating ORAI1. The C-terminal rearrangement of STIM1 does not require a functional CRAC channel, suggesting interaction with ORAI1 as sufficient for this conformational switch. Extended conformations were also engineered by mutations within the first and third coiled-coil domains in the cytosolic portion of STIM1 revealing the involvement of hydrophobic residues in the intramolecular transition. Corresponding full-length STIM1 mutants exhibited enhanced interaction with ORAI1 inducing constitutive CRAC currents, even in the absence of store depletion. We suggest that these mutant STIM1 proteins imitate a physiological activated state, which mimics the intramolecular transition that occurs in native STIM1 upon store depletion.

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Related in: MedlinePlus

Engineering OASF head-to-tail proximity by mutations. (A, B) Localization and calculated NFRET life cell image series of YFP–OASF–CFP wild-type and mutants without (A) or with (B) ORAI1 co-expressed. Calibration bar is 5 μm throughout. (C) Block diagram summarizing NFRET of double-labelled OASF mutants: YFP–OASF–CFP (wild type), YFP–OASF L251S–CFP, YFP–OASF A376K–CFP, YFP–OASF L416S L423S–CFP and YFP–OASF R426L–CFP. (D) Intensity plots representing localization of YFP–OASF–CFP wild-type and mutants without (upper panel) and with (lower panel) ORAI1 in regions close to the plasma membrane as indicated by the dashed line.
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f3: Engineering OASF head-to-tail proximity by mutations. (A, B) Localization and calculated NFRET life cell image series of YFP–OASF–CFP wild-type and mutants without (A) or with (B) ORAI1 co-expressed. Calibration bar is 5 μm throughout. (C) Block diagram summarizing NFRET of double-labelled OASF mutants: YFP–OASF–CFP (wild type), YFP–OASF L251S–CFP, YFP–OASF A376K–CFP, YFP–OASF L416S L423S–CFP and YFP–OASF R426L–CFP. (D) Intensity plots representing localization of YFP–OASF–CFP wild-type and mutants without (upper panel) and with (lower panel) ORAI1 in regions close to the plasma membrane as indicated by the dashed line.

Mentions: STIM1 encodes three putative coiled-coil domains (Hogan et al, 2010) within the cytosolic portion (Supplementary Figure S2A) that might contribute to the OASF conformation via intramolecular interactions. In general, coiled-coil domains are well known for mediating intermolecular as well as intramolecular protein associations via both hydrophobic and electrostatic interactions (Steinmetz et al, 2007; Grigoryan and Keating, 2008; Parry et al, 2008). In an attempt to engineer OASF in its extended conformation, we initially decreased OASF length from its N-terminal side, as CAD/SOAR is primarily devoid of the first coiled-coil domain (aa 233–342). We further mutated various hydrophobic leucines at position a or d within a heptad repeat (Woolfson, 2005) that are highly conserved between various species (Supplementary Figure S3), in an attempt to locally interfere with the putative first coiled-coil structure. Among several constructs (Supplementary Figure S4B), the very N-terminal portion (aa 233–251) appeared most interesting, as its deletion or L to S point mutations therein (L248S, L251S) led to constructs with a substantial reduction of FRET compared with the wild-type sensor (Figure 3A and C; Supplementary Figure S4B). To circumvent the impact of N-terminal truncations on FRET, we focused (below) on the OASF L251S point mutant, which assumed an extended conformation independent of interaction with ORAI1. Several other L to S mutations within the first coiled-coil downstream to aa L251 led to smaller or almost no reduction of FRET (Supplementary Figure S4B) underscoring the importance of the N-terminal region (aa 233–251) of OASF to this conformational transition. Thus, the first, putative coiled-coil domain likely has a role in intramolecular coiled-coil associations within OASF.


STIM1 couples to ORAI1 via an intramolecular transition into an extended conformation.

Muik M, Fahrner M, Schindl R, Stathopulos P, Frischauf I, Derler I, Plenk P, Lackner B, Groschner K, Ikura M, Romanin C - EMBO J. (2011)

Engineering OASF head-to-tail proximity by mutations. (A, B) Localization and calculated NFRET life cell image series of YFP–OASF–CFP wild-type and mutants without (A) or with (B) ORAI1 co-expressed. Calibration bar is 5 μm throughout. (C) Block diagram summarizing NFRET of double-labelled OASF mutants: YFP–OASF–CFP (wild type), YFP–OASF L251S–CFP, YFP–OASF A376K–CFP, YFP–OASF L416S L423S–CFP and YFP–OASF R426L–CFP. (D) Intensity plots representing localization of YFP–OASF–CFP wild-type and mutants without (upper panel) and with (lower panel) ORAI1 in regions close to the plasma membrane as indicated by the dashed line.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3101990&req=5

f3: Engineering OASF head-to-tail proximity by mutations. (A, B) Localization and calculated NFRET life cell image series of YFP–OASF–CFP wild-type and mutants without (A) or with (B) ORAI1 co-expressed. Calibration bar is 5 μm throughout. (C) Block diagram summarizing NFRET of double-labelled OASF mutants: YFP–OASF–CFP (wild type), YFP–OASF L251S–CFP, YFP–OASF A376K–CFP, YFP–OASF L416S L423S–CFP and YFP–OASF R426L–CFP. (D) Intensity plots representing localization of YFP–OASF–CFP wild-type and mutants without (upper panel) and with (lower panel) ORAI1 in regions close to the plasma membrane as indicated by the dashed line.
Mentions: STIM1 encodes three putative coiled-coil domains (Hogan et al, 2010) within the cytosolic portion (Supplementary Figure S2A) that might contribute to the OASF conformation via intramolecular interactions. In general, coiled-coil domains are well known for mediating intermolecular as well as intramolecular protein associations via both hydrophobic and electrostatic interactions (Steinmetz et al, 2007; Grigoryan and Keating, 2008; Parry et al, 2008). In an attempt to engineer OASF in its extended conformation, we initially decreased OASF length from its N-terminal side, as CAD/SOAR is primarily devoid of the first coiled-coil domain (aa 233–342). We further mutated various hydrophobic leucines at position a or d within a heptad repeat (Woolfson, 2005) that are highly conserved between various species (Supplementary Figure S3), in an attempt to locally interfere with the putative first coiled-coil structure. Among several constructs (Supplementary Figure S4B), the very N-terminal portion (aa 233–251) appeared most interesting, as its deletion or L to S point mutations therein (L248S, L251S) led to constructs with a substantial reduction of FRET compared with the wild-type sensor (Figure 3A and C; Supplementary Figure S4B). To circumvent the impact of N-terminal truncations on FRET, we focused (below) on the OASF L251S point mutant, which assumed an extended conformation independent of interaction with ORAI1. Several other L to S mutations within the first coiled-coil downstream to aa L251 led to smaller or almost no reduction of FRET (Supplementary Figure S4B) underscoring the importance of the N-terminal region (aa 233–251) of OASF to this conformational transition. Thus, the first, putative coiled-coil domain likely has a role in intramolecular coiled-coil associations within OASF.

Bottom Line: The C-terminal rearrangement of STIM1 does not require a functional CRAC channel, suggesting interaction with ORAI1 as sufficient for this conformational switch.Corresponding full-length STIM1 mutants exhibited enhanced interaction with ORAI1 inducing constitutive CRAC currents, even in the absence of store depletion.We suggest that these mutant STIM1 proteins imitate a physiological activated state, which mimics the intramolecular transition that occurs in native STIM1 upon store depletion.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biophysics, University of Linz, Linz, Austria.

ABSTRACT
Stromal interaction molecule (STIM1) and ORAI1 are key components of the Ca(2+) release-activated Ca(2+) (CRAC) current having an important role in T-cell activation and mast cell degranulation. CRAC channel activation occurs via physical interaction of ORAI1 with STIM1 when endoplasmic reticulum Ca(2+) stores are depleted. Here we show, utilizing a novel STIM1-derived Förster resonance energy transfer sensor, that the ORAI1 activating small fragment (OASF) undergoes a C-terminal, intramolecular transition into an extended conformation when activating ORAI1. The C-terminal rearrangement of STIM1 does not require a functional CRAC channel, suggesting interaction with ORAI1 as sufficient for this conformational switch. Extended conformations were also engineered by mutations within the first and third coiled-coil domains in the cytosolic portion of STIM1 revealing the involvement of hydrophobic residues in the intramolecular transition. Corresponding full-length STIM1 mutants exhibited enhanced interaction with ORAI1 inducing constitutive CRAC currents, even in the absence of store depletion. We suggest that these mutant STIM1 proteins imitate a physiological activated state, which mimics the intramolecular transition that occurs in native STIM1 upon store depletion.

Show MeSH
Related in: MedlinePlus