Single-Molecule Studies of the ER Calcium Sensor STIM1

Abstract

The ER membrane (EM) protein STIM1 is the ER Ca2+ sensor that initiates store-operated Ca2+ entry through Orai channels in lymphocytes and many other cells. Communication of ER Ca2+ store depletion to Orai channels in the plasma membrane involves a transition of STIM1 dimers from an inactive to an active state, but the conformational changes underlying this transition remain poorly characterized. We have developed an in-vitro assay to study the conformation and dynamics of STIM1 at the single-molecule level. Purified STIM1 cytosolic fragments were labeled at single sites with Alexa555 and Alexa647 by site-directed mutagenesis and cysteine-maleimide chemistry. These served as donor and acceptor dyes for high-resolution intradimer distance measurements using single-molecule FRET. Labeled STIM1 fragments were encapsulated in 100nm diameter, surface-immobilized liposomes and imaged with a prism-based TIRF microscope.We identified three broad regions within the STIM1 fragments, defined by their characteristic FRET signatures. Within the ER-proximal N-terminal region containing the predicted coiled-coil 1 domain (aa274-339), we observed several transiently stable conformational states, ranging from closely apposed (FRET ∼0.8) to widely separated (FRET ∼0). The different FRET states occurred with similar probability, and transitions between them were frequent. In the region 363-449, encompassing the CRAC activation domain (CAD), stable intermediate to high FRET values (0.6-0.9) were dominant, indicating close apposition of paired sites in the dimer. Interestingly, low-FRET states (0.1-0.4) were also observed at several sites, suggesting the existence of an open conformation with relatively low probability. Finally, C-terminal to the CAD (aa512-660) we found stable, broad low-FRET signals, consistent with the predicted unstructured nature of this region. FRET ratios progressively declined from ∼0.2 to 0 towards the C-terminal end, indicating increasing separation. Further development of this approach will enable direct, high-resolution probing of STIM1 activation under controlled conditions.