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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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Dimerization/oligomerization and conformation of OASF and mutants. (A) Block diagram summarizing intermolecular NFRET between double-labelled CFP/CFP and YFP/YFP OASF forms: OASF (wild type), OASF L251S, OASF L416S L423S and OASF R426L. (B) Purity and far-UV CD spectra of OASF-ext (aa 234–491) mutant and wild-type forms. Spectra were acquired at 20°C in 20 mM Tris, 200 mM NaCl, 2 mM DTT, pH 8 using protein concentrations ranging from 0.14 to 0.35 mg ml−1. Protein purity was confirmed using Coomassie-stained SDS–PAGE (inset). (C) SEC with in-line MALS analyses of OASF-ext (aa 234–491) mutant and wild-type forms. SEC experiments were performed at 4°C in 20 mM Tris, 100 mM NaCl, 50 mM L-Arg/L-Glu, 2 mM DTT, pH 8 using 0.85–2.0 mg ml−1 protein.
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f4: Dimerization/oligomerization and conformation of OASF and mutants. (A) Block diagram summarizing intermolecular NFRET between double-labelled CFP/CFP and YFP/YFP OASF forms: OASF (wild type), OASF L251S, OASF L416S L423S and OASF R426L. (B) Purity and far-UV CD spectra of OASF-ext (aa 234–491) mutant and wild-type forms. Spectra were acquired at 20°C in 20 mM Tris, 200 mM NaCl, 2 mM DTT, pH 8 using protein concentrations ranging from 0.14 to 0.35 mg ml−1. Protein purity was confirmed using Coomassie-stained SDS–PAGE (inset). (C) SEC with in-line MALS analyses of OASF-ext (aa 234–491) mutant and wild-type forms. SEC experiments were performed at 4°C in 20 mM Tris, 100 mM NaCl, 50 mM L-Arg/L-Glu, 2 mM DTT, pH 8 using 0.85–2.0 mg ml−1 protein.

Mentions: In summary, the L251S, A376K and L416/423S OASF sensor mutants showed substantial FRET reduction by ∼0.4 compared with the OASF wild-type form, which likely resulted from a pronounced decrease of head-to-tail proximity. Intermolecular FRET measurements suggested comparable degree of dimerization/oligomerization of OASF and mutants (Figure 4A).


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)

Dimerization/oligomerization and conformation of OASF and mutants. (A) Block diagram summarizing intermolecular NFRET between double-labelled CFP/CFP and YFP/YFP OASF forms: OASF (wild type), OASF L251S, OASF L416S L423S and OASF R426L. (B) Purity and far-UV CD spectra of OASF-ext (aa 234–491) mutant and wild-type forms. Spectra were acquired at 20°C in 20 mM Tris, 200 mM NaCl, 2 mM DTT, pH 8 using protein concentrations ranging from 0.14 to 0.35 mg ml−1. Protein purity was confirmed using Coomassie-stained SDS–PAGE (inset). (C) SEC with in-line MALS analyses of OASF-ext (aa 234–491) mutant and wild-type forms. SEC experiments were performed at 4°C in 20 mM Tris, 100 mM NaCl, 50 mM L-Arg/L-Glu, 2 mM DTT, pH 8 using 0.85–2.0 mg ml−1 protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Dimerization/oligomerization and conformation of OASF and mutants. (A) Block diagram summarizing intermolecular NFRET between double-labelled CFP/CFP and YFP/YFP OASF forms: OASF (wild type), OASF L251S, OASF L416S L423S and OASF R426L. (B) Purity and far-UV CD spectra of OASF-ext (aa 234–491) mutant and wild-type forms. Spectra were acquired at 20°C in 20 mM Tris, 200 mM NaCl, 2 mM DTT, pH 8 using protein concentrations ranging from 0.14 to 0.35 mg ml−1. Protein purity was confirmed using Coomassie-stained SDS–PAGE (inset). (C) SEC with in-line MALS analyses of OASF-ext (aa 234–491) mutant and wild-type forms. SEC experiments were performed at 4°C in 20 mM Tris, 100 mM NaCl, 50 mM L-Arg/L-Glu, 2 mM DTT, pH 8 using 0.85–2.0 mg ml−1 protein.
Mentions: In summary, the L251S, A376K and L416/423S OASF sensor mutants showed substantial FRET reduction by ∼0.4 compared with the OASF wild-type form, which likely resulted from a pronounced decrease of head-to-tail proximity. Intermolecular FRET measurements suggested comparable degree of dimerization/oligomerization of OASF and mutants (Figure 4A).

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