Resolving the molecular determinants underlying the conformational stability of STIM2

Authors

  • S Narayanasamy* (Co-first authors)
  • HL Ong*
  • S Choi
  • D Bonhenry
  • M Sallinger
  • PB Stathopulos
  • R Schindl
  • I Ambudkar

Abstract

STIM1 and STIM2 are ER proteins that sense decreases ER-[Ca2+] and activate Orai1 channel resulting in store-operated calcium entry. The Ca2+-sensing N-terminus of these proteins consists of nEF, cEF, and SAM domains. Despite the considerable homology between them, the STIM proteins display distinct Ca2+ affinities and sensitivity to agonist-stimulated ER-Ca2+ depletion. STIM2, with relatively low Ca2+ affinity, appears to be in an “active” state even in absence of substantial ER-Ca2+ depletion. Intriguingly, previous far-UV CD studies have shown that STIM2 conformation is only minimally affected by presence or absence of Ca2+, unlike STIM1 which displays marked conformational changes under these conditions. In this study we have assessed the molecular determinants underlying the conformational stability of STIM2. First, we mutated STIM2-cEF (GV/TF) to enhance the Ca2+-binding affinity of STIM2. Interestingly, STIM2GV/TF displayed Ca2+ affinity higher that STIM2-WT and similar to STIM1. However, MD simulation and far-UV CD studies showed that the mutant was more like STIM2-WT and showed minimal Ca2+-dependent conformational changes. Importantly, we identified a region of close (<4Ao) apposition between nEF and cEF that was very stably maintained. Introduction of negatively charged residues in this region (EEDD) disrupted the conformation with expansion of nEF and cEF domains. The rate and extent of this expansion was increased by an additional mutation (F158A) in the SAM region. Together, our findings suggest that while specific residues in the cEF determine the Ca2+ affinity of STIM2, intramolecular interactions between nEF, cEF, and SAM domains contribute to the conformational stability of the protein.

Scientific Focus Area: Molecular Biology and Biochemistry

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