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Oligomerization and Ca2+/calmodulin control binding of the ER Ca2+-sensors STIM1 and STIM2 to plasma membrane lipids.

Bhardwaj R, Müller HM, Nickel W, Seedorf M - Biosci. Rep. (2013)

Bottom Line: We found that tetramerization of the STIM1 K-rich domain is necessary for efficient binding to PI(4,5)P2-containing PM-like liposomes consistent with an oligomerization-driven STIM1 activation.Concomitant with higher affinity for PM lipids, binding of CaM (calmodulin) inhibited the interaction of the STIM2 K-rich domain with liposomes in a Ca2+ and PI(4,5)P2 concentration-dependent manner.Therefore we suggest that elevated cytosolic Ca2+ concentration down-regulates STIM2-mediated ER-PM contacts via CaM binding.

View Article: PubMed Central - PubMed

Affiliation: *Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, University of Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.

ABSTRACT
Ca2+ (calcium) homoeostasis and signalling rely on physical contacts between Ca2+ sensors in the ER (endoplasmic reticulum) and Ca2+ channels in the PM (plasma membrane). STIM1 (stromal interaction molecule 1) and STIM2 Ca2+ sensors oligomerize upon Ca2+ depletion in the ER lumen, contact phosphoinositides at the PM via their cytosolic lysine (K)-rich domains, and activate Ca2+ channels. Differential sensitivities of STIM1 and STIM2 towards ER luminal Ca2+ have been studied but responses towards elevated cytosolic Ca2+ concentration and the mechanism of lipid binding remain unclear. We found that tetramerization of the STIM1 K-rich domain is necessary for efficient binding to PI(4,5)P2-containing PM-like liposomes consistent with an oligomerization-driven STIM1 activation. In contrast, dimerization of STIM2 K-rich domain was sufficient for lipid binding. Furthermore, the K-rich domain of STIM2, but not of STIM1, forms an amphipathic α-helix. These distinct features of the STIM2 K-rich domain cause an increased affinity for PI(4,5)P2, consistent with the lower activation threshold of STIM2 and a function as regulator of basal Ca2+ levels. Concomitant with higher affinity for PM lipids, binding of CaM (calmodulin) inhibited the interaction of the STIM2 K-rich domain with liposomes in a Ca2+ and PI(4,5)P2 concentration-dependent manner. Therefore we suggest that elevated cytosolic Ca2+ concentration down-regulates STIM2-mediated ER-PM contacts via CaM binding.

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The K-rich domain of STIM2 forms an α-helix and binds PI(4,5)P2 as dimer(A) Alignment of extreme C-termini of STIM proteins from different species. Alignment of K-rich domains from STIM1 in mammals and fishes are shown on the left and STIM2 on the right. Conservation of proline and cysteine residues are highlighted (B) Cartoon of GFP fused to the last 27 residues of STIM2 (GFP–STIM2K) where Cys725 is depicted as grey circle. Coomassie Brilliant Blue stained GFP–STIM2K [WT (wild-type)] and GFP–STIM2K with a C725A mutation (C725A) after overnight dialysis in HK buffer (25 mM Hepes/KOH pH 7.5, 150 mM NaCl) and separation by SDS/PAGE under reducing and non-reducing conditions. (C) Binding of 1 μM reduced GFP and GFP–STIM2C, oxidized and reduced GFP–STIM2K, GFP–STIM2K–C725A dialysed against DTT-free buffer and reduced GFP–STIM2K–C725A to PM-like liposomes with 5 mol% PI(4,5)P2. Binding of oxidized GFP–STIM2K was set to 100. Bars indicate mean±S.D. from at least three experiments and n.s. (not significant) according to student's t test. (D) Cartoon of GFP fused to the last 24 residues of STIM1 (GFP–STIM1K) and positions of the introduced Leu and Cys residues and Pro to Ala mutations. Coomassie Brilliant Blue stained GFP–STIM1K (WT), GFP–STIM1K+LC and GFP–STIM1K+LC–P675A–P682A after overnight dialysis in HK buffer with and without 1 mM DTT and separation by SDS/PAGE under reducing and non-reducing conditions. (E) Binding of 1 μM oxidized GFP–STIM2K, GFP–STIM1K dialysed against DTT-free buffer, reduced GFP–STIM1K, oxidized and reduced GFP–STIM1K+LC, GFP–STIM1K+LC-P675A-P682A and reduced GFP-Zipper-STIM1K to PM-like liposomes with 5 mol% PI(4,5)P2. Binding of 1 μM oxidized GFP–STIM2K was set to 100. Bars indicate means±S.D. from at least three experiments (**P<0.005 according to student's t test). (F) Sequence alignment of peptides of human STIM1 (residues 671–685) and STIM2 (residues 730–746) corresponding to the K-rich domains used for CD. CD spectra of 150 μM peptide in 5 mM KPi buffer pH 7.6 containing 0, 20, 40 and 50% TFE, is shown below the alignment.
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Figure 2: The K-rich domain of STIM2 forms an α-helix and binds PI(4,5)P2 as dimer(A) Alignment of extreme C-termini of STIM proteins from different species. Alignment of K-rich domains from STIM1 in mammals and fishes are shown on the left and STIM2 on the right. Conservation of proline and cysteine residues are highlighted (B) Cartoon of GFP fused to the last 27 residues of STIM2 (GFP–STIM2K) where Cys725 is depicted as grey circle. Coomassie Brilliant Blue stained GFP–STIM2K [WT (wild-type)] and GFP–STIM2K with a C725A mutation (C725A) after overnight dialysis in HK buffer (25 mM Hepes/KOH pH 7.5, 150 mM NaCl) and separation by SDS/PAGE under reducing and non-reducing conditions. (C) Binding of 1 μM reduced GFP and GFP–STIM2C, oxidized and reduced GFP–STIM2K, GFP–STIM2K–C725A dialysed against DTT-free buffer and reduced GFP–STIM2K–C725A to PM-like liposomes with 5 mol% PI(4,5)P2. Binding of oxidized GFP–STIM2K was set to 100. Bars indicate mean±S.D. from at least three experiments and n.s. (not significant) according to student's t test. (D) Cartoon of GFP fused to the last 24 residues of STIM1 (GFP–STIM1K) and positions of the introduced Leu and Cys residues and Pro to Ala mutations. Coomassie Brilliant Blue stained GFP–STIM1K (WT), GFP–STIM1K+LC and GFP–STIM1K+LC–P675A–P682A after overnight dialysis in HK buffer with and without 1 mM DTT and separation by SDS/PAGE under reducing and non-reducing conditions. (E) Binding of 1 μM oxidized GFP–STIM2K, GFP–STIM1K dialysed against DTT-free buffer, reduced GFP–STIM1K, oxidized and reduced GFP–STIM1K+LC, GFP–STIM1K+LC-P675A-P682A and reduced GFP-Zipper-STIM1K to PM-like liposomes with 5 mol% PI(4,5)P2. Binding of 1 μM oxidized GFP–STIM2K was set to 100. Bars indicate means±S.D. from at least three experiments (**P<0.005 according to student's t test). (F) Sequence alignment of peptides of human STIM1 (residues 671–685) and STIM2 (residues 730–746) corresponding to the K-rich domains used for CD. CD spectra of 150 μM peptide in 5 mM KPi buffer pH 7.6 containing 0, 20, 40 and 50% TFE, is shown below the alignment.

Mentions: Our previous studies, however, showed that the isolated K-rich domain of STIM2 (GFP–STIM2K), lacking its CC domains, can bind PI(4,5)P2-containing liposomes [6]. Both K-rich domains of STIM1 and STIM2 comprise a similar number of positively charged residues (eight and nine) and this is unlikely to explain the difference in lipid binding. To unravel this difference, we compared the sequences of STIM1 and STIM2 K-rich domains from different species. Most C-termini of STIM proteins in metazoa contain a K-rich sequence (Figure 2A). We also noted that unlike in STIM1, STIM2 evolved a conserved cysteine residue, which is located directly upstream of the K-rich domain. This residue may dimerize the K-rich domain during our purification procedure using dialysis under non-reducing conditions [6]. In order to test whether dimerization of STIM2 K-rich domain causes the increased affinity to PI(4,5)P2 liposomes, we performed overnight dialysis of GFP–STIM2K in a non-reducing (DTT-free) buffer. This led to formation of a DTT-reducible dimer, as validated by SDS/PAGE (Figure 2B). Dimerization is strictly dependent on Cys725 as mutation to alanine completely abolished dimer formation (Figure 2B). The STIM2 K-rich domain dimer was sufficient for binding to PI(4,5)P2-containing PM-like liposomes and showed no significant difference in binding compared with tetrameric STIM2 CTD in presence of DTT (Figure 2C). Only the oxidized GFP–STIM2K dimer but neither the reduced monomer nor the Cys-725-Ala mutant bound PI(4,5)P2 liposomes (Figure 2C).


Oligomerization and Ca2+/calmodulin control binding of the ER Ca2+-sensors STIM1 and STIM2 to plasma membrane lipids.

Bhardwaj R, Müller HM, Nickel W, Seedorf M - Biosci. Rep. (2013)

The K-rich domain of STIM2 forms an α-helix and binds PI(4,5)P2 as dimer(A) Alignment of extreme C-termini of STIM proteins from different species. Alignment of K-rich domains from STIM1 in mammals and fishes are shown on the left and STIM2 on the right. Conservation of proline and cysteine residues are highlighted (B) Cartoon of GFP fused to the last 27 residues of STIM2 (GFP–STIM2K) where Cys725 is depicted as grey circle. Coomassie Brilliant Blue stained GFP–STIM2K [WT (wild-type)] and GFP–STIM2K with a C725A mutation (C725A) after overnight dialysis in HK buffer (25 mM Hepes/KOH pH 7.5, 150 mM NaCl) and separation by SDS/PAGE under reducing and non-reducing conditions. (C) Binding of 1 μM reduced GFP and GFP–STIM2C, oxidized and reduced GFP–STIM2K, GFP–STIM2K–C725A dialysed against DTT-free buffer and reduced GFP–STIM2K–C725A to PM-like liposomes with 5 mol% PI(4,5)P2. Binding of oxidized GFP–STIM2K was set to 100. Bars indicate mean±S.D. from at least three experiments and n.s. (not significant) according to student's t test. (D) Cartoon of GFP fused to the last 24 residues of STIM1 (GFP–STIM1K) and positions of the introduced Leu and Cys residues and Pro to Ala mutations. Coomassie Brilliant Blue stained GFP–STIM1K (WT), GFP–STIM1K+LC and GFP–STIM1K+LC–P675A–P682A after overnight dialysis in HK buffer with and without 1 mM DTT and separation by SDS/PAGE under reducing and non-reducing conditions. (E) Binding of 1 μM oxidized GFP–STIM2K, GFP–STIM1K dialysed against DTT-free buffer, reduced GFP–STIM1K, oxidized and reduced GFP–STIM1K+LC, GFP–STIM1K+LC-P675A-P682A and reduced GFP-Zipper-STIM1K to PM-like liposomes with 5 mol% PI(4,5)P2. Binding of 1 μM oxidized GFP–STIM2K was set to 100. Bars indicate means±S.D. from at least three experiments (**P<0.005 according to student's t test). (F) Sequence alignment of peptides of human STIM1 (residues 671–685) and STIM2 (residues 730–746) corresponding to the K-rich domains used for CD. CD spectra of 150 μM peptide in 5 mM KPi buffer pH 7.6 containing 0, 20, 40 and 50% TFE, is shown below the alignment.
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Show All Figures
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Figure 2: The K-rich domain of STIM2 forms an α-helix and binds PI(4,5)P2 as dimer(A) Alignment of extreme C-termini of STIM proteins from different species. Alignment of K-rich domains from STIM1 in mammals and fishes are shown on the left and STIM2 on the right. Conservation of proline and cysteine residues are highlighted (B) Cartoon of GFP fused to the last 27 residues of STIM2 (GFP–STIM2K) where Cys725 is depicted as grey circle. Coomassie Brilliant Blue stained GFP–STIM2K [WT (wild-type)] and GFP–STIM2K with a C725A mutation (C725A) after overnight dialysis in HK buffer (25 mM Hepes/KOH pH 7.5, 150 mM NaCl) and separation by SDS/PAGE under reducing and non-reducing conditions. (C) Binding of 1 μM reduced GFP and GFP–STIM2C, oxidized and reduced GFP–STIM2K, GFP–STIM2K–C725A dialysed against DTT-free buffer and reduced GFP–STIM2K–C725A to PM-like liposomes with 5 mol% PI(4,5)P2. Binding of oxidized GFP–STIM2K was set to 100. Bars indicate mean±S.D. from at least three experiments and n.s. (not significant) according to student's t test. (D) Cartoon of GFP fused to the last 24 residues of STIM1 (GFP–STIM1K) and positions of the introduced Leu and Cys residues and Pro to Ala mutations. Coomassie Brilliant Blue stained GFP–STIM1K (WT), GFP–STIM1K+LC and GFP–STIM1K+LC–P675A–P682A after overnight dialysis in HK buffer with and without 1 mM DTT and separation by SDS/PAGE under reducing and non-reducing conditions. (E) Binding of 1 μM oxidized GFP–STIM2K, GFP–STIM1K dialysed against DTT-free buffer, reduced GFP–STIM1K, oxidized and reduced GFP–STIM1K+LC, GFP–STIM1K+LC-P675A-P682A and reduced GFP-Zipper-STIM1K to PM-like liposomes with 5 mol% PI(4,5)P2. Binding of 1 μM oxidized GFP–STIM2K was set to 100. Bars indicate means±S.D. from at least three experiments (**P<0.005 according to student's t test). (F) Sequence alignment of peptides of human STIM1 (residues 671–685) and STIM2 (residues 730–746) corresponding to the K-rich domains used for CD. CD spectra of 150 μM peptide in 5 mM KPi buffer pH 7.6 containing 0, 20, 40 and 50% TFE, is shown below the alignment.
Mentions: Our previous studies, however, showed that the isolated K-rich domain of STIM2 (GFP–STIM2K), lacking its CC domains, can bind PI(4,5)P2-containing liposomes [6]. Both K-rich domains of STIM1 and STIM2 comprise a similar number of positively charged residues (eight and nine) and this is unlikely to explain the difference in lipid binding. To unravel this difference, we compared the sequences of STIM1 and STIM2 K-rich domains from different species. Most C-termini of STIM proteins in metazoa contain a K-rich sequence (Figure 2A). We also noted that unlike in STIM1, STIM2 evolved a conserved cysteine residue, which is located directly upstream of the K-rich domain. This residue may dimerize the K-rich domain during our purification procedure using dialysis under non-reducing conditions [6]. In order to test whether dimerization of STIM2 K-rich domain causes the increased affinity to PI(4,5)P2 liposomes, we performed overnight dialysis of GFP–STIM2K in a non-reducing (DTT-free) buffer. This led to formation of a DTT-reducible dimer, as validated by SDS/PAGE (Figure 2B). Dimerization is strictly dependent on Cys725 as mutation to alanine completely abolished dimer formation (Figure 2B). The STIM2 K-rich domain dimer was sufficient for binding to PI(4,5)P2-containing PM-like liposomes and showed no significant difference in binding compared with tetrameric STIM2 CTD in presence of DTT (Figure 2C). Only the oxidized GFP–STIM2K dimer but neither the reduced monomer nor the Cys-725-Ala mutant bound PI(4,5)P2 liposomes (Figure 2C).

Bottom Line: We found that tetramerization of the STIM1 K-rich domain is necessary for efficient binding to PI(4,5)P2-containing PM-like liposomes consistent with an oligomerization-driven STIM1 activation.Concomitant with higher affinity for PM lipids, binding of CaM (calmodulin) inhibited the interaction of the STIM2 K-rich domain with liposomes in a Ca2+ and PI(4,5)P2 concentration-dependent manner.Therefore we suggest that elevated cytosolic Ca2+ concentration down-regulates STIM2-mediated ER-PM contacts via CaM binding.

View Article: PubMed Central - PubMed

Affiliation: *Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, University of Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.

ABSTRACT
Ca2+ (calcium) homoeostasis and signalling rely on physical contacts between Ca2+ sensors in the ER (endoplasmic reticulum) and Ca2+ channels in the PM (plasma membrane). STIM1 (stromal interaction molecule 1) and STIM2 Ca2+ sensors oligomerize upon Ca2+ depletion in the ER lumen, contact phosphoinositides at the PM via their cytosolic lysine (K)-rich domains, and activate Ca2+ channels. Differential sensitivities of STIM1 and STIM2 towards ER luminal Ca2+ have been studied but responses towards elevated cytosolic Ca2+ concentration and the mechanism of lipid binding remain unclear. We found that tetramerization of the STIM1 K-rich domain is necessary for efficient binding to PI(4,5)P2-containing PM-like liposomes consistent with an oligomerization-driven STIM1 activation. In contrast, dimerization of STIM2 K-rich domain was sufficient for lipid binding. Furthermore, the K-rich domain of STIM2, but not of STIM1, forms an amphipathic α-helix. These distinct features of the STIM2 K-rich domain cause an increased affinity for PI(4,5)P2, consistent with the lower activation threshold of STIM2 and a function as regulator of basal Ca2+ levels. Concomitant with higher affinity for PM lipids, binding of CaM (calmodulin) inhibited the interaction of the STIM2 K-rich domain with liposomes in a Ca2+ and PI(4,5)P2 concentration-dependent manner. Therefore we suggest that elevated cytosolic Ca2+ concentration down-regulates STIM2-mediated ER-PM contacts via CaM binding.

Show MeSH
Related in: MedlinePlus