Coordination of a Single Calcium Ion in the EF-hand Maintains the Off State of the Stromal Interaction Molecule Luminal Domain.

Store operated calcium (Ca²⁺) entry (SOCE) is the process whereby endoplasmic reticulum (ER) Ca²⁺ store depletion causes Orai1-composed Ca²⁺ channels on the plasma membrane (PM) to open, mediating a rise in cytosolic Ca²⁺ levels. Stromal interaction molecules (STIMs) are the proteins that directly sense ER Ca²⁺ content and gate Orai1 channels due to store depletion. The trigger for STIM activation is Ca²⁺ unbinding from the ER lumen-oriented domains, which consist of a nonconserved amino (N) terminal region and EF-hand and sterile α motif (SAM) domains (EF-SAM), highly conserved from humans to Caenorhabditis elegans. Solution NMR structures of the human EF-SAM domains have been determined at high Ca²⁺ concentrations; however, no direct structural view of the Ca²⁺ binding mode has been elucidated. Further, no atomic resolution data currently exists on EF-SAM at low Ca²⁺ levels. Here, we determined the X-ray crystal structure of the C. elegans STIM luminal domain, revealing that EF-SAM binds a single Ca²⁺ ion with pentagonal bipyramidal geometry and an ancillary α-helix formed by the N-terminal region acts as a brace to stabilize EF-SAM. Using solution NMR, we observed EF-hand domain unfolding and a conformational exchange between folded and unfolded states involving the ancillary α-helix and the canonical EF-hand in low Ca²⁺. Remarkably, we also detected an α-helix (+Ca²⁺) to β-strand (-Ca²⁺) transition at the terminal SAM domain α-helix. Collectively, our analyses indicate that one canonically bound Ca²⁺ ion is sufficient to stabilize the quiescent luminal domain structure, precluding unfolding, conformational exchange, and secondary structure transformation.

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