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.

The STIM1-binding site nexus remotely controls Orai1 channel gating

“…1a)791819. The C-terminal M4-ext is connected to M4 by a conserved flexible ‘hinge' (SHK; residues S263, H264 and K265)131820. Immediately upstream of the hinge, residues V262 and L261 closely approach the M3 helix, with L261 in close contact with L174 and A175 (Fig.…”

The STIM-Orai Pathway: Conformational Coupling Between STIM and Orai in the Activation of Store-Operated Ca2+ Entry

“…The nexus is a discrete sequence and is composed of two fundamental regions. The first is the “hinge” domain (Ser-263, His-264, Lys-265) (Hou et al 2012; Palty et al 2015; Tirado-Lee et al 2015) and, the second, the “hinge plate” (Leu-261, Val-262). Specifically, the residues of the hinge plate appear to be the hydrophobic attachment to TM3 and are the key transducers of the STIM1-binding signal.…”

“…Previous groups have studied the hinge region by substituting with proline or cross-linking with cysteine through a redox reaction. These two alterations will lock the hinge and prevent the binding of STIM1 and Ca 2+ entry through Orai1 (Palty et al 2015; Tirado-Lee et al 2015). However, no group has studied the “hinge plate” region, which based on the crystal structure of Orai1 appears to be the main contact point between TM4 and TM3, specifically at residues Leu-261 from TM4 and Leu-174 from TM3.…”

“…These extensions appear to be held together through hydrophobic interactions between Leu-273 and Leu-276, which are required for STIM1 binding (Fig. 5.5) (Tirado-Lee et al 2015). We propose that STIM1 binding to the Orai1 C-terminus acts by prying the TM4-extensions apart, perhaps through interactions with the Phe-394 residue in the SOAR active site that we described earlier.…”

The STIM-Orai Pathway: Orai, the Pore-Forming Subunit of the CRAC Channel

“…The solution NMR structure of a fragment of STIM1 complexed with Orai1(272–292) illustrates one way in which a STIM1 dimer could bind to a pair of adjacent Orai1 C-terminal helices (Stathopulos et al 2013). Other experiments have pointed to an alternative binding model in which Orai C-terminal helices interact individually with STIM1 (Hou et al 2012; Zhou et al 2015, Tirado-Lee et al 2015, Palty et al 2015). It is, of course, conceivable that both modes of binding occur during the physiological interaction of STIM1 with the channel.…”