Calcium sensing by the STIM1 ER-luminal domain.

Stromal interaction molecule 1 (STIM1) monitors ER-luminal Ca levels to maintain cellular Ca balance and to support Ca signalling. The prevailing view has been that STIM1 senses reduced ER Ca through dissociation of bound Ca from a single EF-hand site, which triggers a dramatic loss of secondary structure and dimerization of the STIM1 luminal domain. Here we find that the STIM1 luminal domain has 5-6 Ca-binding sites, that binding at these sites is energetically coupled to binding at the EF-hand site, and that Ca dissociation controls a switch to a second structured conformation of the luminal domain rather than protein unfolding.

Coiled-Coil Formation Conveys a STIM1 Signal from ER Lumen to Cytoplasm.

STIM1 and STIM2 are endoplasmic reticulum (ER) membrane proteins that sense decreases in ER-luminal free Ca and, through a conformational change in the STIM cytoplasmic domain, control gating of the plasma membrane Ca channel ORAI1. To determine how STIM1 conveys a signal from the ER lumen to the cytoplasm, we studied the Ca-dependent conformational change of engineered STIM1 proteins in isolated ER membranes and, in parallel, physiological activation of these proteins in cells. We find that conserved "sentinel" features of the CC1 region help to prevent activation while Ca is bound to STIM ER-luminal domains.

TMEM110 regulates the maintenance and remodeling of mammalian ER-plasma membrane junctions competent for STIM-ORAI signaling.

The stromal interaction molecule (STIM)-ORAI calcium release-activated calcium modulator (ORAI) pathway controls store-dependent calcium entry, a major mechanism of physiological calcium signaling in mammalian cells. The core elements of the pathway are the regulatory protein STIM1, located in the endoplasmic reticulum (ER) membrane, the calcium channel ORAI1 in the plasma membrane, and sites of close contact between the ER and the plasma membrane that permit the two proteins to interact. Research on calcium signaling has centered on STIM1, ORAI1, and a few proteins that directly modulate STIM-ORAI function.

Secretagogin, a hexa EF-hand calcium-binding protein: high level bacterial overexpression, one-step purification and properties.

Secretagogin (SCGN), a hexa EF-hand calcium-binding protein, is highly expressed in the endocrine cells (especially in pancreatic islets) and in restricted neuronal sub-populations, albeit at comparatively low level. Since SCGN is predicted to be a potential neuroendocrine marker in carcinoid tumors of lung and gastrointestinal tract, it is of paramount importance to understand the features of this protein in different environment for assigning its crucial functions in different tissues and under pathophysiological conditions. To score out the limitation of protein for in vitro studies, we report a one-step, high purity and high level bacterial purification of secretagogin by refolding from the inclusion bodies yielding about 40mg protein per litre of bacterial culture.

Liaison between myristoylation and cryptic EF-hand motif confers Ca(2+) sensitivity to neuronal calcium sensor-1.

Many members of the neuronal calcium sensor (NCS) protein family have a striking coexistence of two characteristics, that is, N-myristoylation and the cryptic EF-1 motif. We investigated the rationale behind this correlation in neuronal calcium sensor-1 (NCS-1) by restoring Ca(2+) binding ability of the disabled EF-1 loop by appropriate mutations. The concurrence of canonical EF-1 and N-myristoylation considerably decreased the overall Ca(2+) affinity, conformational flexibility, and functional activation of downstream effecter molecules (i.