Abstract
Stromal interaction molecule 1 (STIM1) has recently been identified as a key player in store-operated Ca2+ entry. Endoplasmic reticulum (ER) luminal Ca2+ depletion results in STIM1 redistribution from ER membrane homogeneity to distinctly localized aggregates near the plasma membrane; these changes precede and are linked to cytoplasmic Ca2+ influx via Ca2+ release-activated channels (CRACs). The molecular mechanisms initiating ER STIM1 redistribution and plasma membrane CRAC activity are not well understood. We recombinantly expressed the Ca2+-sensing region of STIM1 consisting of the EF-hand together with the sterile alpha-motif (SAM) domain (EF-SAM) to investigate its Ca2+-related conformational and biochemical features. We demonstrate that Ca2+-loaded EF-SAM (holo) contains high alpha-helicity, whereas EF-SAM in the absence of Ca2+ (apo) is much less compact. Accordingly, the melting temperature (Tm) of the holoform is approximately 25 degrees C higher than apoform; heat and urea-derived thermodynamic parameters indicate a Ca2+-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 Ca2+ binding affinity of EF-SAM is relatively low with an apparent dissociation constant (Kd) of approximately 0.2-0.6 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 Ca2+ affinity of EF-SAM is reconciled with the confirmed role of STIM1 as an ER Ca2+ sensor.
Highlights
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]
We show that holoEF-SAM exists as a monomer, whereas apoEFSAM 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
The results demonstrate that EF-SAM binds Ca2ϩ with an affinity that can be reconciled with physiological endoplasmic reticulum (ER) luminal and extracellular Ca2ϩ concentrations
Summary
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]. SOCE is the process whereby modest ER Ca2ϩ store depletion leads to plasma membrane (PM) Ca2ϩ release-activated channel (CRAC) activation, providing a sustained Ca2ϩ elevation in the cytoplasm from extracellular sources and refilling the ER luminal Ca2ϩ stores [6]. Interfering and small inhibiting RNA studies have independently implicated stromal interaction molecule-1 (STIM1) as the likely Ca2ϩ 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 [3,4,5]. The N-terminal regions of STIM1 include a signal peptide, putative EF-hand motif, and predicted sterile ␣-motif (SAM) domain. This study provides a mechanistic perspective on recent observations made on changes in physical STIM1 distribution associated with Ca2ϩ fluctuations in cell culture
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