Stromal interaction molecule 1 (STIM1) has recently been identified as a key player in store-operated Ca2+ entry. Endoplasmic reticulum (ER) luminal Ca2+ depletion results in STIM1 redistribution from ER membrane homogeneity to distinctly localized aggregates near the plasma membrane; these changes precede and are linked to cytoplasmic Ca2+ influx via Ca2+ release-activated channels (CRACs). The molecular mechanisms initiating ER STIM1 redistribution and plasma membrane CRAC activity are not well understood. We recombinantly expressed the Ca2+-sensing region of STIM1 consisting of the EF-hand together with the sterile alpha-motif (SAM) domain (EF-SAM) to investigate its Ca2+-related conformational and biochemical features. We demonstrate that Ca2+-loaded EF-SAM (holo) contains high alpha-helicity, whereas EF-SAM in the absence of Ca2+ (apo) is much less compact. Accordingly, the melting temperature (Tm) of the holoform is approximately 25 degrees C higher than apoform; heat and urea-derived thermodynamic parameters indicate a Ca2+-induced stabilization of 3.2 kcal mol(-1). We show that holoEF-SAM exists as a monomer, whereas apoEF-SAM readily forms a dimer and/or oligomer, and that oligomer to monomer transitions and vice versa are at least in part mediated by changes in surface hydrophobicity. Additionally, we find that the Ca2+ binding affinity of EF-SAM is relatively low with an apparent dissociation constant (Kd) of approximately 0.2-0.6 mM and a binding stoichiometry of 1. Our results suggest that EF-SAM actively participates in and is the likely the molecular trigger initiating STIM1 punctae formation via large conformational changes. The low Ca2+ affinity of EF-SAM is reconciled with the confirmed role of STIM1 as an ER Ca2+ sensor.

Significance: KRAS (Kirsten rat sarcoma viral oncogene homolog) is frequently mutated in pancreatic, colon, and lung tumors, which predicts poor clinical outcome, whereas germ-line mutations are associated with developmental disorders, including Noonan syndrome. Although K-RAS is an attractive anticancer target, no clinically successful inhibitors are available. Most disease-associated mutations elevate the activated GTP-bound form of KRAS; however, some remain unexplained. KRAS signals from cellular membranes; however, our studies revealed that its association with the membrane surface sequesters its binding site for effector proteins, hampering signaling. Some disease-associated KRAS mutations disrupt this autoinhibition, identifying a new gain-of-function mechanism and explaining how certain Noonan syndrome mutations activate K-RAS signaling. Importantly, these findings open new avenues for therapeutic strategies to target oncogenic K-RAS through stabilizing autoinhibitory interactions with the membrane.

In immune cells, CRAC channels in the plasma membrane regulate store-operated Ca2+ entry in response to STIM1, a sensor protein located in the endoplasmic reticulum (ER) membrane. New biophysical assays show how dimerization of STIM1's ER-luminal domains causes extension of its cytoplasmic domains toward the plasma membrane to contact the ORAI pore and activate the channel, revealing the structural dynamics of this Ca2+-signaling mechanism. Physiological Ca2+ signaling in T lymphocytes and other cells depends on the STIM-ORAI pathway of store-operated Ca2+ entry. STIM1 and STIM2 are Ca2+ sensors in the endoplasmic reticulum (ER) membrane, with ER-luminal domains that monitor cellular Ca2+ stores and cytoplasmic domains that gate ORAI channels in the plasma membrane. The STIM ER-luminal domain dimerizes or oligomerizes upon dissociation of Ca2+, but the mechanism transmitting activation to the STIM cytoplasmic domain was previously undefined. Using Tb3+-acceptor energy transfer, we show that dimerization of STIM1 ER-luminal domains causes an extensive conformational change in mouse STIM1 cytoplasmic domains. The conformational change, triggered by apposition of the predicted coiled-coil 1 (CC1) regions, releases the ORAI-activating domains from their interaction with the CC1 regions and allows physical extension of the STIM1 cytoplasmic domain across the gap between ER and plasma membrane and communication with ORAI channels.