Molecular dynamics simulations reveal the hidden EF-hand of EF-SAM as a possible key thermal sensor for STIM1 activation by temperature

Intracellular calcium signaling is essential for many cellular processes, including store-operated Ca²⁺ entry (SOCE), which is initiated by stromal interaction molecule 1 (STIM1) detecting endoplasmic reticulum (ER) Ca²⁺ depletion. STIM1 is also activated by temperature independent of ER Ca²⁺ depletion. Here we provide evidence, from advanced molecular dynamics simulations, that EF-SAM may act as a true temperature sensor for STIM1, with the prompt and extended unfolding of the hidden EF-hand subdomain (hEF) even at slightly elevated temperatures, exposing a highly conserved hydrophobic Phe108. Our study also suggests an interplay between Ca²⁺ and temperature sensing, as both, the canonical EF-hand subdomain (cEF) and the hidden EF-hand subdomain (hEF), exhibit much higher thermal stability in the Ca²⁺-loaded form compared to the Ca²⁺-free form. The SAM domain, surprisingly, displays high thermal stability compared to the EF-hands and may act as a stabilizer for the latter. We propose a modular architecture for the EF-hand-SAM domain of STIM1 composed of a thermal sensor (hEF), a Ca²⁺ sensor (cEF), and a stabilizing domain (SAM). Our findings provide important insights into the mechanism of temperature-dependent regulation of STIM1, which has broad implications for understanding the role of temperature in cellular physiology.