Stored Ca2+ Depletion-induced Oligomerization of Stromal Interaction Molecule 1 (STIM1) via the EF-SAM Region

Abstract

-loaded EF-SAM (holo) contains high ␣-helicity, whereas EF-SAM in the absence of Ca 2؉ (apo) is much less compact. Accordingly, the melting temperature (T m ) of the holoform is ϳ25°C higher than apoform; heat and urea-derived thermodynamic parameters indicate a Ca 2؉ -induced stabilization of 3.2 kcal mol ؊1 . We show that holoEF-SAM exists as a monomer, whereas apoEF-SAM readily forms a dimer and/or oligomer, and that oligomer to monomer transitions and vice versa are at least in part mediated by changes in surface hydrophobicity. Additionally, we find that the Ca 2؉ binding affinity of EF-SAM is relatively low with an apparent dissociation constant (K d ) of ϳ0.2-0.6 mM and a binding stoichiometry of 1. Our results suggest that EF-SAM actively participates in and is the likely the molecular trigger initiating STIM1 punctae formation via large conformational changes. The low Ca 2؉ affinity of EF-SAM is reconciled with the confirmed role of STIM1 as an ER Ca 2؉ sensor.Calcium is a fundamental signaling messenger in every eukaryotic cell, regulating a multitude of diverse and kinetically distinct cellular phenomena including gene transcription, protein folding, protein degradation, apoptosis, necrosis, and exocytosis, to name a few (1). The endoplasmic reticulum (ER) 4 is a network of folded membranes that extends through the cytoplasm to the nuclear envelope of eukaryotes. The ER membranes surround an inner cavity, the lumen, that is critical to the function of the ER as a Ca 2ϩ signaling organelle (2). Because vital Ca 2ϩ -dependent processes are associated with the ER, it is essential that changes in luminal Ca 2ϩ levels do not adversely affect these phenomena. Eukaryotes have evolved store-operated Ca 2ϩ entry (SOCE), also termed capacitive Ca 2ϩ entry, as a major Ca 2ϩ entry pathway in electrochemically non-excitable cells (3-5). SOCE is the process whereby modest ER Ca 2ϩ store depletion leads to plasma membrane (PM) Ca 2ϩ release-activated channel (CRAC) activation, providing a sustained Ca 2ϩ elevation in the cytoplasm from extracellular sources and ultimately refilling the ER luminal Ca 2ϩ stores (6). Until recently, the molecular link between ER Ca 2ϩ efflux and extracellular influx was not known. However, interfering and small inhibiting RNA studies have independently implicated stromal interaction molecule-1 (STIM1) as the likely Ca 2ϩ sensor in the ER (7,8). This single-pass, type I transmembrane protein of 685 amino acids has been found localized on both the plasma and ER membranes (3-5). The N-terminal regions of STIM1 include a signal peptide, putative EF-hand motif, and predicted sterile ␣-motif (SAM) domain. The cytosolic C-terminal region consists of two coiled-coil domains, a Pro/Ser-rich region, and a Lys-rich region (supplemental Fig. 1A) (9 -11). The putative EF-hand of STIM1 strongly aligns with the helix-loop-helix consensus sequence of this Ca 2ϩ binding motif (supplemental Fig. 1B). The EF-hand in STIM1 is somewhat unorthodox because it is seemingly unpaired, whereas Ca 2ϩ sensor pr...

Voltage Gating at the Selectivity Filter of the Ca2+ Release-activated Ca2+ Channel Induced by Mutation of the Orai1 Protein

“…2). Based on our data and that of others, we propose that the oligomerization of STIM1 within the ER membrane is dominated by the intermolecular interactions of the STIM1 EF-hand sterile ␣-motif domain region (19), whereas the interactions of ER-STIM1 with the PM proteins are structurally supported by the coiled-coil region of STIM1. Such interactions likely involve homomultimerization of ER-STIM1 with the PM-STIM1 (Fig.…”

“…Based on the protein structure and several studies on STIM1, we hypothesize that two distinct types of interactions are likely important in the function of STIM1. First, homotypic interactions likely mediate the aggregation of the molecule into puncta after Ca 2ϩ store depletion (19). Recent studies indicate that an N-terminal fragment of STIM1 that contains the sterile ␣-motif and the EF-hand domains is able to undergo self-association upon removal of Ca 2ϩ (19).…”

“…First, homotypic interactions likely mediate the aggregation of the molecule into puncta after Ca 2ϩ store depletion (19). Recent studies indicate that an N-terminal fragment of STIM1 that contains the sterile ␣-motif and the EF-hand domains is able to undergo self-association upon removal of Ca 2ϩ (19). These results suggest that the ER luminal N terminus of STIM1 mediates the punctal self-assembly of STIM1 when the ER Ca 2ϩ is depleted.…”

Calcium signaling in lymphocytes

“…The propensity of STIM1 to aggregate when its EF-hands are not bound by Ca 2+ can be demonstrated using just a short recombinant N-terminal fragment of STIM1, containing only the EF-hands and the adjacent SAM (sterile α-motif) domain [56,57]. Once formed, the STIM1 clusters preferentially localize to sites of ER-plasma membrane apposition and colocalize partially with clusters of ORAI1 [55,58, [61,62].…”

Alternative splicing converts STIM2 from an activator to an inhibitor of store-operated calcium channels

“…The stromal interaction molecule (STIM) family of ER Ca 2+ sensors (STIM1 and STIM2; Liou et al, 2005;Roos et al, 2005;Zhang et al, 2005) and the Orai Ca 2+ channels (Orai1, 2, and 3; Feske et al, 2006;Vig et al, 2006) are key molecular mediators of SOCE (Cahalan, 2009;Hogan et al, 2010;Lewis, 2011). Store depletion triggers oligomerization (Stathopulos et al, 2006;Covington et al, 2010) and conformational rearrangements of STIM proteins (Muik et al, 2011). These rearrangements expose the C-terminal polybasic domain, which interacts with phosphatidylinositol 4,5-bisphosphate in the plasma membrane (PM) and drives STIM accumulation at ER-PM junctions (Wu et al, 2006;Liou et al, 2007;Ercan et al, 2009;Park et al, 2009).…”