Conformational changes in the Orai1 C-terminus evoked by STIM1 binding

Leidamarie Tirado-Lee, Megumi Yamashita, Murali Prakriya

Research output: Contribution to journalArticle

31 Scopus citations

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 crosslinking 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.

Original languageEnglish (US)
Article numbere0128622
JournalPloS one
Volume10
Issue number6
DOIs
StatePublished - Jun 2 2015

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

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