Premium
A single EF‐hand isolated from STIM1 forms dimer in the absence and presence of Ca 2+
Author(s) -
Huang Yun,
Zhou Yubin,
Wong HingCheung,
Chen Yanyi,
Chen Yan,
Wang Siming,
Castiblanco Adriana,
Liu Aimin,
Yang Jenny J.
Publication year - 2009
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2009.07240.x
Subject(s) - endoplasmic reticulum , stim1 , orai1 , dimer , ef hand , chemistry , dissociation constant , biophysics , crystallography , protein subunit , mutant , stereochemistry , calcium , biochemistry , calcium binding protein , biology , receptor , organic chemistry , gene
Stromal interaction molecule 1 (STIM1) is responsible for activating the Ca 2+ release‐activated Ca 2+ (CRAC) channel by first sensing the changes in Ca 2+ concentration in the endoplasmic reticulum ([Ca 2+ ] 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 Ca 2+ with a dissociation constant at a level comparable with [Ca 2+ ] ER (512 ± 15 μ m ). The binding of Ca 2+ resulted in a more compact conformation of the engineered protein. Our results also showed that D to A mutations at Ca 2+ ‐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 Ca 2+ concentration in the endoplasmic reticulum ([Ca 2+ ] ER ) and resulted in constitutive punctae formation and Ca 2+ influx. In addition, the grafted single EF‐hand motif formed a dimer regardless of the presence of Ca 2+ , 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 [Ca 2+ ] ER .