title: Conformational Changes in the Orai1 C-Terminus Evoked by STIM1 Binding.
abstract: Store-operated CRAC channels regulate a wide range of cellular functions including gene expression, chemotaxis, and proliferation. CRAC channels consist of two components: the Orai proteins (Orai1-3), which form the ion-selective pore, and STIM proteins (STIM1-2), which form the endoplasmic reticulum (ER) Ca2+ sensors. Activation of CRAC channels is initiated by the migration of STIM1 to the ER-plasma membrane (PM) junctions, where it directly interacts with Orai1 to open the Ca2+-selective pores of the CRAC channels. The recent elucidation of the Drosophila Orai structure revealed a hexameric channel wherein the C-terminal helices of adjacent Orai subunits associate in an anti-parallel orientation. This association is maintained by hydrophobic interactions between the Drosophila equivalents of human Orai1 residues L273 and L276. Here, we used mutagenesis and chemical cross-linking to assess the nature and extent of conformational changes in the self-associated Orai1 C-termini during STIM1 binding. We find that linking the anti-parallel coiled-coils of the adjacent Orai1 C-termini through disulfide cross-links diminishes STIM1-Orai1 interaction, as assessed by FRET. Conversely, prior binding of STIM1 to the Orai1 C-terminus impairs cross-linking of the Orai1 C-termini. Mutational analysis indicated that a bend of the Orai1 helix located upstream of the self-associated coils (formed by the amino acid sequence SHK) establishes an appropriate orientation of the Orai1 C-termini that is required for STIM1 binding. Together, our results support a model wherein the self-associated Orai1 C-termini rearrange modestly to accommodate STIM1 binding.
authors.affiliation: Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States of America.
authors.forename: Leidamarie
authors.initials: L
authors.lastname: Tirado-Lee
authors.affiliation: Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States of America.
authors.forename: Megumi
authors.initials: M
authors.lastname: Yamashita
authors.affiliation: Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States of America.
authors.forename: Murali
authors.initials: M
authors.lastname: Prakriya
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020013:Calcium Signaling; D002462:Cell Membrane / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism*; D000071740:ORAI1 Protein; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits; D017386:Sequence Homology, Amino Acid; D000071737:Stromal Interaction Molecule 1
chemical_list: D015220:Calcium Channels; D029721:Drosophila Proteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; D021122:Protein Subunits; C103530:STIM1 protein, human; C502662:Stim protein, Drosophila; D000071737:Stromal Interaction Molecule 1; C510747:olf186-F protein, Drosophila