title: Stored Ca2+ depletion-induced oligomerization of stromal interaction molecule 1 (STIM1) via the EF-SAM region: An initiation mechanism for capacitive Ca2+ entry.
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.
authors.affiliation: Division of Signaling Biology and Department of Medical Biophysics, Ontario Cancer Institute and University of Toronto, Toronto M5G 1L7, Ontario, Canada.
authors.forename: Peter B
authors.initials: PB
authors.lastname: Stathopulos
authors.forename: Guang-Yao
authors.initials: GY
authors.lastname: Li
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Plevin
authors.forename: James B
authors.initials: JB
authors.lastname: Ames
authors.forename: Mitsuhiko
authors.initials: M
authors.lastname: Ikura
mesh_terms: D002118:Calcium / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000502:physiology*; D008954:Models, Biological; D009363:Neoplasm Proteins / Q000737:chemistry / Q000502:physiology*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D000071737:Stromal Interaction Molecule 1; D013696:Temperature
chemical_list: D008565:Membrane Proteins; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D002118:Calcium