Conformational Changes in the Orai1 C-Terminus Evoked by STIM1 Binding
Fig 5
A conserved motif in the bend region of the Orai1 C-terminus is required for STIM1 binding.
(A) Sequence alignment of the Orai1 C-terminus from various species shows a conserved stretch of amino acids spanning residues 260–268 (human Orai1) that include the SHK residues. (B) Close-up of Drosophila Orai1 C-terminus with the conserved ‘SHK’ bend highlighted in green. Because the side chain for K265 was not modeled into the Drosophila structure, we used the PyMol mutagenesis wizard to generate three different potential rotomer conformations of this residue. (C) Summary of E-FRET for mutants of S263 and K265 Orai1-YFP with CFP-CAD. E-FRET values for each cell were averaged from three frames. Data points are mean ± SEM for 15–42 cells (*** p ≤ 0.001; * p ≤ 0.05). (D) Summary of E-FRET for mutation of S263 in Orai1-YFP to the equivalent residues in Orai2 (arginine, R) and Orai3 (alanine, A) with CFP-CAD. Data points are mean ± SEM for 16–19 cells. (E) Close-up of TM4a ‘SHK’ hairpin bend in one monomer. Highlighted in green are residues S263 and Q271, which were mutated to cysteine. Formation of an intra-monomer disulfide bond between these residues is predicted to restrict conformational changes at the bend. (F) Summary of E-FRET between the S263/Q271C Orai-YFP bend double mutant and CFP-CAD, in the presence of diamide or BMS. E-FRET values for each cell were averaged from three frames. Data points are mean ± SEM for 15–32 cells (*** p ≤ 0.001; ** p ≤ 0.01).