Conformational Changes in the Orai1 C-Terminus Evoked by STIM1 Binding

PLoS One. 2015 Jun 2;10(6):e0128622. doi: 10.1371/journal.pone.0128622. eCollection 2015.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Blotting, Western
  • Calcium Channels / chemistry*
  • Calcium Channels / genetics
  • Calcium Channels / metabolism
  • Calcium Signaling*
  • Cell Membrane / metabolism
  • Drosophila Proteins / chemistry*
  • Drosophila Proteins / genetics
  • Drosophila Proteins / metabolism*
  • Endoplasmic Reticulum / metabolism
  • HEK293 Cells
  • Humans
  • Ion Channel Gating / physiology
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation / genetics
  • Neoplasm Proteins / chemistry
  • Neoplasm Proteins / metabolism*
  • ORAI1 Protein
  • Protein Binding
  • Protein Conformation
  • Protein Subunits
  • Sequence Homology, Amino Acid
  • Stromal Interaction Molecule 1


  • Calcium Channels
  • Drosophila Proteins
  • Membrane Proteins
  • Neoplasm Proteins
  • ORAI1 Protein
  • ORAI1 protein, human
  • Protein Subunits
  • STIM1 protein, human
  • Stim protein, Drosophila
  • Stromal Interaction Molecule 1
  • olf186-F protein, Drosophila