STIM1 couples to ORAI1 via an intramolecular transition into an extended conformation
2011; Springer Nature; Volume: 30; Issue: 9 Linguagem: Inglês
10.1038/emboj.2011.79
ISSN1460-2075
AutoresMartin Muik, Marc Fahrner, Rainer Schindl, Peter B. Stathopulos, Irene Frischauf, Isabella Derler, Peter Plenk, Barbara Lackner, Klaus Groschner, Mitsuhiko Ikura, Christoph Romanin,
Tópico(s)Ion channel regulation and function
ResumoArticle22 March 2011Open Access STIM1 couples to ORAI1 via an intramolecular transition into an extended conformation Martin Muik Martin Muik Institute of Biophysics, University of Linz, Linz, Austria Search for more papers by this author Marc Fahrner Marc Fahrner Institute of Biophysics, University of Linz, Linz, Austria Search for more papers by this author Rainer Schindl Rainer Schindl Institute of Biophysics, University of Linz, Linz, Austria Search for more papers by this author Peter Stathopulos Peter Stathopulos Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Irene Frischauf Irene Frischauf Institute of Biophysics, University of Linz, Linz, Austria Search for more papers by this author Isabella Derler Isabella Derler Institute of Biophysics, University of Linz, Linz, Austria Search for more papers by this author Peter Plenk Peter Plenk Institute of Biophysics, University of Linz, Linz, Austria Search for more papers by this author Barbara Lackner Barbara Lackner Institute of Biophysics, University of Linz, Linz, Austria Search for more papers by this author Klaus Groschner Klaus Groschner Institute of Pharmaceutical Sciences, University of Graz, Graz, Austria Search for more papers by this author Mitsuhiko Ikura Mitsuhiko Ikura Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Christoph Romanin Corresponding Author Christoph Romanin Institute of Biophysics, University of Linz, Linz, Austria Search for more papers by this author Martin Muik Martin Muik Institute of Biophysics, University of Linz, Linz, Austria Search for more papers by this author Marc Fahrner Marc Fahrner Institute of Biophysics, University of Linz, Linz, Austria Search for more papers by this author Rainer Schindl Rainer Schindl Institute of Biophysics, University of Linz, Linz, Austria Search for more papers by this author Peter Stathopulos Peter Stathopulos Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Irene Frischauf Irene Frischauf Institute of Biophysics, University of Linz, Linz, Austria Search for more papers by this author Isabella Derler Isabella Derler Institute of Biophysics, University of Linz, Linz, Austria Search for more papers by this author Peter Plenk Peter Plenk Institute of Biophysics, University of Linz, Linz, Austria Search for more papers by this author Barbara Lackner Barbara Lackner Institute of Biophysics, University of Linz, Linz, Austria Search for more papers by this author Klaus Groschner Klaus Groschner Institute of Pharmaceutical Sciences, University of Graz, Graz, Austria Search for more papers by this author Mitsuhiko Ikura Mitsuhiko Ikura Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Christoph Romanin Corresponding Author Christoph Romanin Institute of Biophysics, University of Linz, Linz, Austria Search for more papers by this author Author Information Martin Muik1,‡, Marc Fahrner1,‡, Rainer Schindl1,‡, Peter Stathopulos2, Irene Frischauf1, Isabella Derler1, Peter Plenk1, Barbara Lackner1, Klaus Groschner3, Mitsuhiko Ikura2 and Christoph Romanin 1 1Institute of Biophysics, University of Linz, Linz, Austria 2Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada 3Institute of Pharmaceutical Sciences, University of Graz, Graz, Austria ‡These authors contributed equally to this work *Corresponding author. Institute of Biophysics, University of Linz, 4040 Linz, Austria. Tel.: +43 732 2468 9272; Fax: +43 732 2468 9280; E-mail: [email protected] The EMBO Journal (2011)30:1678-1689https://doi.org/10.1038/emboj.2011.79 There is a Have you seen? (May 2011) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Stromal interaction molecule (STIM1) and ORAI1 are key components of the Ca2+ release-activated Ca2+ (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 Ca2+ 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. Introduction Calcium signalling in the cytosol of excitable and non-excitable cells is of crucial importance. It triggers both short-term responses like secretion, muscle contraction or metabolism and also long-term regulation including transcription, cell growth and apoptosis (Berridge et al, 2003). A major calcium pathway is mediated by store-operated channels (SOCs). The endoplasmic reticulum (ER) calcium stores are depleted upon binding of inositol-1,4,5-triphosphate (IP3) to their receptors, resulting in the activation of the plasma membrane Ca2+ release-activated Ca2+ (CRAC) channels (Parekh and Putney, 2005). CRAC channels are characterized by a high Ca2+ selectivity and very low single channel conductance (Hoth and Penner, 1992; Parekh and Putney, 2005). Their activation enhances cytosolic Ca2+ levels and thereby stimulates gene expression via the nuclear factor of activated T cells, resulting in cytokine secretion in the early stages of immune responses (Feske, 2007; Oh-hora and Rao, 2008). A systematic genetic screen by RNA interference has discovered the stromal interaction molecule (STIM1) and ORAI1 (also termed CRACM1) as the main molecular components of CRAC channels (Liou et al, 2005; Roos et al, 2005; Feske et al, 2006; Zhang et al, 2006; Vig et al, 2006b; Hogan et al, 2010). STIM1 has been identified as the ER-located Ca2+ sensor (Liou et al, 2005; Roos et al, 2005), which senses the luminal Ca2+ content by its N-terminal EF hand. ORAI1 is a Ca2+ selective channel located in the plasma membrane with four transmembrane segments and cytosolic N- and C-terminal strands (Prakriya et al, 2006; Yeromin et al, 2006; Vig et al, 2006b; Schindl et al, 2008). A mutation within ORAI1, that is R91W, leading to a non-functional channel has been directly linked to severe combined immune deficiency (SCID) (Feske et al, 2006). At resting state, STIM1 is uniformly distributed throughout the ER membrane. Store depletion triggers STIM1 multimer formation. These aggregates then translocate into puncta close to the plasma membrane (Liou et al, 2005, 2007; Zhang et al, 2005; Baba et al, 2006; Luik et al, 2006; Mercer et al, 2006; Soboloff et al, 2006; Wu et al, 2006; Xu et al, 2006), thereby activating ORAI1/CRAC channels. Besides an EF-hand pair, the luminal N-terminus of STIM1 further contains a sterile-α motif, all these domains are required for multimerization (Stathopulos et al, 2008, 2009). Following a single transmembrane helix, the cytoplasmic C terminus includes three coiled-coil domains (Hogan et al, 2010), which overlap with an ezrin–radixin–moesin (ERM)-like domain, a serine/proline- and a lysine-rich segment (Liou et al, 2005; Baba et al, 2006; Huang et al, 2006; Smyth et al, 2006) (see also Supplementary Figure S4A). The CAD/SOAR domain encompasses roughly aa 340–448 including the second and third coiled-coil domain and represents the smallest STIM1 C-terminal fragment that couples to and activates ORAI1 channels (Park et al, 2009; Yuan et al, 2009). An interaction between CAD and both the N- and C-terminal regions of ORAI1 has been determined by in vitro pull-down experiments (Park et al, 2009). We and others have visualized coupling between STIM1 and ORAI1 in store-depleted cells by Förster resonance energy transfer (FRET) microscopy (Barr et al, 2008; Muik et al, 2008; Navarro-Borelly et al, 2008). The cytosolic C-terminal portion of ORAI1 that contains a putative coiled-coil domain couples with STIM1 (Muik et al, 2008; Frischauf et al, 2009). In this study, we designed a STIM1 conformational sensor that demonstrates an intramolecular transition into an extended conformation when binding to ORAI1. Engineering extended conformations via mutations in coiled-coil domains facilitated STIM1 coupling to ORAI1 probably by alterations of coiled-coil intramolecular interactions. The STIM1 conformational sensor further revealed novel characteristics of the SCID-linked ORAI1–R91W mutant and the CRAC modifier 2-aminoethoxy-diphenyl-borate (2-APB) in their interactions with STIM1. Results A STIM1-derived conformational sensor The conformational choreography that evokes activation of ORAI channels via their interaction with STIM1 is unclear. While intermolecular FRET measurements within an assembly of CFP- and YFP-tagged ORAI1 proteins have been carried out (Navarro-Borelly et al, 2008), intramolecular FRET within one ORAI1 protein might provide a more direct read-out of conformational rearrangements that occur upon coupling with STIM1. However, N- and C-terminally double-labelled ORAI1 proteins were not applicable by reason of significantly reduced plasma membrane localization (data not shown). Therefore, we focused on the development of a STIM1-derived conformational sensor that might allow for monitoring of intramolecular rearrangements within the STIM1 cytosolic portion when interacting with ORAI1. As the STIM1 C-terminus acts as a surrogate for full-length STIM1 (Huang et al, 2006; Muik et al, 2008), we initially started to generate a variety of double-labelled STIM1 C-terminal constructs of decreasing length (Figure 1) in an attempt to optimize sensor features for reporting conformational rearrangements relevant for the STIM1/ORAI coupling machinery. Focus was placed on constructs that (i) contained at least the minimal regions previously identified for the interaction with ORAI1 (Muik et al, 2009; Park et al, 2009; Yuan et al, 2009) and (ii) were still functional both for the coupling to as well as activation of ORAI1 channels. All constructs included the first coiled-coil region in addition to the CAD/SOAR domain. Expression of these double-labelled constructs in HEK 293 cells revealed a range of FRET values between ∼0.9 and ∼0.2 (Figure 1A). While showing a remarkably high maximum (∼0.9) around our previously reported ORAI1 activating small fragment (OASF; aa 233–474), C-terminal extension up to the wild-type length (aa 685) resulted in a gradual decrease of FRET. Interestingly, fragments shorter than OASF also exhibited an attenuation of FRET (Figure 1A), indicating that construct length is not the only factor determining FRET. Double-labelled fragments aa 233–420/430 are most likely inactive (Zhang et al, 2008; Muik et al, 2009; Park et al, 2009; Yuan et al, 2009), while 233–450 or 233–474 (OASF) and larger fragments are proposed as sufficient for interaction and ORAI1 current activation (Muik et al, 2009). In this study we focused on the double-labelled aa 233–474 fragment, as the former tends to form large clusters without ORAI1 co-expression (Muik et al, 2009; Yuan et al, 2009). Indeed, double-labelled YFP–STIM1–233–474–CFP (termed YFP–OASF–CFP) allowed for robust constitutive activation of ORAI1-derived currents with CRAC-like biophysical characteristics (Figure 1B). The current densities obtained with the double-labelled OASF sensor were somewhat smaller than those of its single-labelled form (Muik et al, 2009) pointing to a slightly reduced activation capacity and/or affinity for ORAI1. The functionality of this conformational STIM1 sensor constitutes a powerful tool to monitor intramolecular rearrangements within OASF upon binding to ORAI1 (see below). Figure 1.Designing a STIM1-derived conformational sensor. (A) Block diagram summarizing intermolecular/intramolecular NFRET of double-labelled YFP–STIM1–CFP fragments: 233–420, 233–430, 233–450, 233–474 (OASF), 233–485, 233–535 and 233–685 (complete STIM1 C-terminus). (B) Time course of constitutive whole-cell inward currents at −86 mV of HEK293 cells expressing YFP–OASF–CFP with ORAI1 (upper panel) and respective I/V curve taken at t=0 s (lower panel). (C) Localization and calculated NFRET life cell image series of selected STIM1 fragments: 233–420, 233–474 (OASF), 233–535 and 233–685. Calibration bar is 5 μm throughout. (D) The YFP–OASF–CFP FRET sensor detected by western blot with an anti-GFP antibody when expressed in HEK293 cells. (E) A cartoon indicating the intramolecular/intermolecular FRET of OASF labelled either with YFP/CFP (left) or CFP/CFP, YFP/YFP (right). Block diagram comparing intermolecular with intramolecular NFRET of double-labelled STIM1 OASF fragments as depicted in the upper panel. Download figure Download PowerPoint This double-labelled YFP–OASF–CFP construct when expressed alone in HEK 293 cells mainly exhibited a uniform, cytosolic distribution and yielded remarkably high FRET (Figure 1A and C), reaching ∼0.9, much higher than ∼0.2 typically found in our previous experiments with single-labelled constructs detecting OASF oligomerization (Muik et al, 2009). YFP–OASF–CFP proteins were detected by an anti-GFP antibody as a single band in western blot corresponding to the complete sensor form without any smaller, cleaved fragments (Figure 1D). The high FRET value of YFP–OASF–CFP might result from both intramolecular and intermolecular proximity of fluorophores, representing a head-to-tail orientation and dimerization/oligomerization (Muik et al, 2009), respectively (Figure 1E). In an attempt to roughly estimate intermolecular FRET, we generated additional constructs that carried identical labels (either CFP or YFP) on both N- and C-termini giving rise only to intermolecular but not intramolecular FRET (Figure 1E). Co-expressed CFP–OASF–CFP and YFP–OASF–YFP revealed smaller FRET values of ∼0.18 (Figure 1E). Thus, the high FRET observed with the YFP–OASF–CFP conformational sensor is suggested to reflect primarily intramolecular rather than intermolecular interactions and assumedly arises from a head-to-tail proximity of fluorophores within OASF (see Figure 1E). Conformational coupling of OASF sensor to ORAI1 The cytosolic portion of STIM1 interacts with both ORAI1 N- and C-termini (Park et al, 2009). The multiple interaction sites leading to ORAI1 activation might involve a conformational rearrangement within STIM1. The YFP–OASF–CFP conformational sensor enabled us for the first time to address this question. Its expression together with unlabelled ORAI1 in HEK293 cells led to a clear redistribution of the OASF sensor with partial plasma membrane as well as cytosolic targeting. The OASF FRET sensor exhibited clearly stronger membrane targeting than the double-labelled whole STIM1 C-terminus (233–685) consistent with its higher affinity to ORAI1, which was abolished with the shorter 233–430 fragment as evident from density profiles (Supplementary Figure S1). YFP–OASF–CFP yielded higher FRET in the cytosol, while significantly lower FRET was obtained with the fraction of the sensor that was targeted close to the plasma membrane where ORAI1 is located (Figure 2A). This decrease of FRET was not due to a diminished intermolecular FRET, as dimerization/oligomerization of OASF when coupled to ORAI1 slightly enhances FRET as previously shown (Muik et al, 2009) for single-labelled and uniformly double-labelled constructs (Supplementary Figure S2A and B). Hence, our data suggest a reduction of intramolecular FRET by a rearrangement within YFP–OASF–CFP into an extended head-to-tail configuration upon its coupling to ORAI1, although an additional change in fluorophore orientation affecting FRET cannot be excluded. This decrease of FRET from the OASF conformational sensor was similarly observed upon co-expression with the non-conducting (Prakriya et al, 2006; Yeromin et al, 2006; Vig et al, 2006a) ORAI1–E106Q mutant (Figure 2B), indicating that it was not directly linked to Ca2+ entry or caused by increases in submembrane intracellular Ca2+ concentrations. Thus, the intramolecular transition to the extended conformation apparently resulted from OASF coupling with ORAI1 upstream of CRAC channel opening and Ca2+ entry. The ORAI1–L273S mutant that exhibits disrupted communication with STIM1 (Muik et al, 2008) consistently failed to interact with OASF conformational sensor, displaying a rather uniform, high FRET that is similar both in cytosolic and plasma membrane adjacent regions (Figure 2C). The extended conformation of OASF when coupled to ORAI1 may reflect a specific, intramolecular transition possibly exposing the minimal region, that is CAD/SOAR (Park et al, 2009; Yuan et al, 2009), essential for this interaction with and/or gating of the ORAI1 channel (Park et al, 2009). Figure 2.OASF sensor coupling to ORAI1. (Right panel) Localization and calculated NFRET life cell image series of HEK293 cells expressing YFP–OASF–CFP and (A) ORAI1, (B) ORAI1E106Q (C) ORAI1L273S. Calibration bar is 5 μm throughout. Magnified section as indicated by the white box highlights the decrease of FRET in regions of the plasma membrane compared with the cytosol in (A, B). (Left panel) Respective block diagram of separately calculated NFRET for regions including the plasma membrane (within the two yellow borders) and the cytosol (within white border). The 'plasma membrane' was assumed as 1.5–2 μm of the edge of the cell image. Download figure Download PowerPoint Engineering head-to-tail proximity of OASF 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. Figure 3.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. Download figure Download PowerPoint To reveal an involvement of the second coiled-coil domain in the head-to-tail proximity of OASF, we engineered L373S, L373S A376S, A376K hydrophobic mutations (Frischauf et al, 2009; Covington et al, 2010) to analogously interfere with the putative coiled-coil structure. Additionally, this region has been suggested (Frischauf et al, 2009; Calloway et al, 2010; Covington et al, 2010) to encompass the STIM1-binding site for ORAI1, as the above mutations interfered with STIM1 coupling to ORAI1 (Frischauf et al, 2009; Covington et al, 2010). All of these YFP–OASF–CFP sensor mutants exhibited a significant reduction of FRET compared with wild type, with the A376K mutant being most pronounced (Supplementary Figure S4B; Figure 3A and C). These data suggest a contribution of the second coiled-coil domain in controlling intramolecular transitions in addition to its role as potential binding site for ORAI1. The C-terminal segment of OASF around aa 420–430 might also have a role in the head-to-tail proximity based on the substantial reduction of FRET upon its deletion (see Figure 1A). In an attempt to alternatively affect head-to-tail proximity of OASF, which might be governed by intramolecular coiled-coil interactions, we aimed to potentially disrupt (L416S, V419S, L423S) or enhance (R426L) the third putative coiled-coil domain (Hogan et al, 2010) by introducing L/V to S or R to L mutations in this region (Gruber et al, 2006). All four residues are highly conserved between various species (Supplementary Figure S3). The former mutants indeed showed a significant attenuation of FRET (Supplementary Figure S4B) with the YFP–OASF L416S L423S–CFP double mutant revealing the most pronounced reduction, while the R426L OASF sensor mutant displayed a significantly higher FRET than wild type (Figure 3A and C; Supplementary Figure S4), implicating the third coiled-coil region in mediating OASF head-to-tail proximity. 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). Figure 4.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. Download figure Download PowerPoint In vitro analyses of purified OASF wild-type and mutant forms provided further evidence for distinct conformations with no change in the oligomerization state (Figure 4B and C). An extended version of OASF (OASF-ext), encompassing aa 234–491 was used in the in vitro analyses because this protein was less susceptible to degradation in Escherichia coli cells. Wild-type, L251S, L416S and L423S OASF-ext proteins were attainable at >95% purity (Figure 4B, inset). All four of the recombinant proteins showed a high α-helicity, assessed by far-UV circular dichroism (CD), typical of coiled-coil motifs. Interestingly, all three mutant forms exhibited more pronounced negative ellipticity (i.e. see 208 and 222 nm) compared with wild type (Figure 4B). These spectral changes probably reflected a conformational change rather than an increased α-helical content, since the thermal melts of the mutant proteins, measured at 222 nm, did not show an enhanced stability expected to accompany increased levels of secondary structure compared with wild type (Supplementary Figure S5). A similar phenomenon occurs with calmodulin, which displays enhanced negative ellipticity in the far-UV CD spectra in response to Ca2+ binding (Martin and Bayley, 1986) without a change in helical content, but rather a conformational rearrangement of the secondary structure elements in three-dimensional space (Finn et al, 1995). Consistent with a mutation-induced conformational transition, all three mutant proteins exhibited a shorter elution time than the wild-type form in size exclusion chromatography (SEC) experiments (Figure 4C). The shorter elution times are not due to a significant change in quaternary structure, since the in-line multiangle light scattering (MALS) analyses suggested dimer molecular weights for mutant and wild-type proteins (Figure 4C). The shorter elution times suggested an extended conformation for the OASF-ext mutants compared with the wild-type form in line with the FRET measurements (Figure 3A). Thus, based on our mutation data, all three coil-coiled regions within the cytosolic portion of STIM1 have a role in mediating the intramolecular head-to-tail proximity of OASF. Coupling of OASF mutants to ORAI1 A decreased YFP–OASF–CFP FRET might reflect an intramolecular transition to an extended conformation with a potential exposure of the CAD/SOAR domain. This concept was tested by co-expressing ORAI1 with L251S, A376K or L416/423S YFP–OASF–CFP mutants that exhibited the most pronounced reduction of FRET compared with the wild-type sensor in the absence of ORAI1. Co-expression of ORAI1 together with the L251S and L416/423S OASF mutants (Figure 3B) revealed clearly stronger membrane localization than that obtained with wild-type OASF sensor as evident from respective intensity profiles (Figure 3D). Hence, a more extended OASF conformation might allow for enhanced binding to ORAI1. The A376K OASF mutant behaved differently, however, exhibiting a reduced FRET but failing to interact with ORAI1 (Figure 3B–D). Previous studies focused on the second coiled-coil domain as a potential site for interaction with ORAI1 C-terminus (Frischauf et al, 2009; Calloway et al, 2010; Covington et al, 2010). This interaction may have been impaired by the A376K mutation despite the extended OASF conformation. The R426L OASF mutant failed to interact with ORAI1 (Figure 3C and D), suggesting that a sequentially more canonical third coiled-coil domain interfered with the extended conformation potentially required for coupling to ORAI1. Hence, mutations within the first and third coiled-coil domains designed to potentially destabilize intramolecular coiled-coil interactions promoted switching of OASF into an extended conformation, this in turn facilitated interaction with ORAI1 probably by enhanced exposure of the CAD/SOAR domain. Coupling of OASF to ORAI1–R91W The ORAI1–R91W mutant linked to SCID represents a non-functional CRAC channel due to a defect in gating/permeation rather than in interaction with full-length STIM1 (Navarro-Borelly et al, 2008; Derler et al, 2009). However, previous FRET microscopy studies show that while the STIM1–ORAI1–R91W interaction is preserved, it is somewhat attenuated (Muik et al, 2008). Surprisingly, co-expression of ORAI1–R91W with wild-type YFP–OASF–CFP revealed no clear evidence for an interaction (Figure 5A). On the other hand, the OASF L251S and L416/423S mutants that showed enhanced interaction with wild-type ORAI1 were capable of coupling to ORAI1–R91W (Figure 5B and C). As expected, they failed to induce an ORAI1–R91W current (data not shown), consistent with a profound ORAI1 gating defect. The density profiles of OASF L251S and L416/423S mutants co-expressed with ORAI1–R91W (Figure 5B and C) suggested a decrease in the affinity for their interaction when compared with profiles of the OASF mutants with wild-type ORAI1 (Figure 3D), consistent with the reduced but detectable interaction with full-length STIM1 (Derler et al, 2009). Hence, the ORAI1–R91W mutant appears deficient in the ability to switch OASF into its extended conformation via interaction. To evaluate the degree of this deficiency, we tested the V419S OASF mutant that showed only slight attenuation of FRET compared with wild-type OASF when expressed in the absence of ORAI1 (Supplementary Figure S4B). The V419S sensor mutant was indeed able to interact with the ORAI1–R91W mutant, and this interaction was accompanied by a substantial decrease of FRET close to the plasma membrane (Figure 5D), consistent with full-length STIM1 observations (Muik et al, 2008; Derler et al, 2009). The ability of the ORAI1–R91W mutant to drive the V419S OASF mutant into a more extended conformation m
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