A single EF‐hand isolated from STIM1 forms dimer in the absence and presence of Ca2

Stromal interaction molecule 1 (STIM1) is responsible for activating the Ca2+ release‐activated Ca2+ (CRAC) channel by first sensing the changes in Ca2+ concentration in the endoplasmic reticulum ([Ca2+]ER) via its luminal canonical EF‐hand motif and subsequently oligomerizing to interact with the CRAC channel pore‐forming subunit Orai1. In this work, we applied a grafting approach to obtain the intrinsic metal‐binding affinity of the isolated EF‐hand of STIM1, and further investigated its oligomeric state using pulsed‐field gradient NMR and size‐exclusion chromatography. The canonical EF‐hand bound Ca2+ with a dissociation constant at a level comparable with [Ca2+]ER (512 ± 15 μm). The binding of Ca2+ resulted in a more compact conformation of the engineered protein. Our results also showed that D to A mutations at Ca2+‐coordinating loop positions 1 and 3 of the EF‐hand from STIM1 led to a 15‐fold decrease in the metal‐binding affinity, which explains why this mutant was insensitive to changes in Ca2+ concentration in the endoplasmic reticulum ([Ca2+]ER) and resulted in constitutive punctae formation and Ca2+ influx. In addition, the grafted single EF‐hand motif formed a dimer regardless of the presence of Ca2+, which conforms to the EF‐hand paring paradigm. These data indicate that the STIM1 canonical EF‐hand motif tends to dimerize for functionality in solution and is responsible for sensing changes in [Ca2+]ER.