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.
Highlights
Calcium (Ca2+) is a ubiquitous second messenger involved in the regulation of a wide assortment of cellular functions including gene expression, proliferation, and chemotaxis [1]
We found that the L273C and L276C single mutants expressed well in the plasma membrane (Fig 2A) and interacted with STIM1 at levels similar to WT Orai1 as assessed by FRET (Fig 2B–2D)
One proposed model hypothesizes that the anti-parallel coiled-coil observed in the crystal structure dissociate and the individual Orai Ctermini extend into the cytosol to interact with STIM1 [24]
Summary
Calcium (Ca2+) is a ubiquitous second messenger involved in the regulation of a wide assortment of cellular functions including gene expression, proliferation, and chemotaxis [1]. Using a combination of mutational analysis and cross-linking of a highly conserved SHK bend motif, upstream of the anti-parallel cross-over, we found that the bend is a crucial structural feature that plays an important role in setting up the Orai C-termini in the proper orientation to accommodate STIM1 binding. These results suggest that the coiled-coil motif seen in the crystal structure likely represents a quiescent state with only modest conformational changes needed for STIM binding
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