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

Abstract

Stromal interaction molecule 1 (STIM1) has recently been identified as a key player in store-operated Ca 2 entry. Endo-plasmic reticulum (ER) luminal Ca 2 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 Ca 2 influx via Ca 2 release-activated channels (CRACs). The molecular mechanisms initiating ER STIM1 redistribution and plasma membrane CRAC activity are not well understood. We recombi-nantly expressed the Ca 2-sensing region of STIM1 consisting of the EF-hand together with the sterile-motif (SAM) domain (EF-SAM) to investigate its Ca 2-related conformational and biochemical features. We demonstrate that Ca 2-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 25 °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 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 Ca 2 affinity of EF-SAM is reconciled with the confirmed role of STIM1 as an ER Ca 2 sensor.