Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 281, Issue 47, Pages 35855-35862Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M608247200
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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 (T-m) of the holoform is similar to 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 (K-d) of similar to 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.
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