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. Author manuscript; available in PMC: 2019 Jan 2.
Published in final edited form as: Cell Rep. 2018 Jan 2;22(1):72–83. doi: 10.1016/j.celrep.2017.12.030

Figure 5. Crosslinking of STIM1 juxtamembrane residues.

Figure 5

A, Abbreviated alignment of STIM sequences from varied species. Further examples are shown in Figure S5. Residues are shown in black if COILS (input matrix MTIDK, using weights, window length 14) calculates a coiled-coil probability greater than 0.5. Other residues are shown in grey. Boxed residues are assigned to core a or d positions by COILS. The brown highlight indicates a break in the predicted heptad repeat that is conserved across these species. The species represented are Nematostella vectensis, starlet sea anemone; Lingula anatina, a lingulid brachiopod; Drosophila melanogaster, common fruit fly; Daphnia pulex, a water flea; Strigamia maritima, a centipede; Strongylocentrotus purpuratus, purple sea urchin; Homo sapiens, human. B, Crosslinking of STIM1 with single cysteine replacements at residues 237, 241, or 244. The STIM1 dimer band is marked with an arrow. Crosslinking at these specific positions was tested because residues 241 and 244 align with predicted coiled-coil core residues of STIM1 orthologues across a broad range of species (Figure S5), and the spacing of residue 237 from these predicted core positions is compatible with its placement in a core position. The residue corresponding to S237 is, in fact, predicted to be a core residue in a few species, but the conservation of a polar serine in most species and its proximity to the transmembrane segment render coiled-coil prediction uncertain. Data from three biological replicates. C, Upper, STIM1 sequence from residues 214–248. Closely packed transmembrane helix residues are highlighted with grey boxes, and juxtamembrane residues that crosslink are highlighted in red. There is a shift in the heptad repeat of helix-helix packing centered on positions 233 and 234. Hatched shading indicates that the spacing of the highlighted residue is compatible with either adjacent heptad assignment. Lower, Either residue 233 or residue 234 alone, as a core residue, could support continuation of an imperfect coiled coil across the region of ambiguity. See also Figures S5, S6, and S7.