Abstract
Stromal interaction molecule 1 (STIM1) monitors ER-luminal Ca2+ levels to maintain cellular Ca2+ balance and to support Ca2+ signalling. The prevailing view has been that STIM1 senses reduced ER Ca2+ through dissociation of bound Ca2+ 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 Ca2+-binding sites, that binding at these sites is energetically coupled to binding at the EF-hand site, and that Ca2+ dissociation controls a switch to a second structured conformation of the luminal domain rather than protein unfolding. Importantly, the other luminal-domain Ca2+-binding sites interact with the EF-hand site to control physiological activation of STIM1 in cells. These findings fundamentally revise our understanding of physiological Ca2+ sensing by STIM1, and highlight molecular mechanisms that govern the Ca2+ threshold for activation and the steep Ca2+ concentration dependence.