A single EF-hand isolated from STIM1 forms dimer in the absence and presence of Ca2+.
A single EF-hand isolated from STIM1 forms dimer in the absence and presence of Ca2+.
复制标题
DOI：
10.1111/j.1742-4658.2009.07240.x
复制
发表时间：
2009-10
期刊：
影响因子：
--
通讯作者：
Yang JJ
中科院分区：
文献类型：
--
作者：
Huang Y;Zhou Y;Wong HC;Chen Y;Chen Y;Wang S;Castiblanco A;Liu A;Yang JJ
基质相互作用分子1（Stromal interaction molecule 1，STIM 1）通过首先经由其管腔经典EF-手形基序（luminal canonical EF-hand motif）感知内质网（[Ca 2 +]ER）中Ca 2+浓度的变化，随后寡聚化以与CRAC通道孔形成亚基Orai 1相互作用，负责激活Ca 2+释放激活的Ca 2+（CRAC）通道。在这项工作中，我们采用接枝的方法来获得固有的金属结合亲和力的隔离EF手的STIM 1，并进一步研究其低聚状态，使用脉冲场梯度NMR和尺寸排阻色谱。经典EF-手结合Ca 2+，解离常数与[Ca 2 +]ER（512 ± 15 μM）相当。Ca ~（2+）的结合导致工程蛋白的构象更紧凑。我们的研究结果还表明，D到A的突变在Ca 2 +-协调环位置1和3的EF-手从STIM 1导致金属结合亲和力降低了15倍，这解释了为什么这个突变体是不敏感的内质网（[Ca 2 +]ER）中的Ca 2+浓度的变化，并导致组成性斑点的形成和Ca 2+内流。此外，接枝的单个EF-手基序形成二聚体，而不管Ca 2+的存在，这符合EF-手配对范例。这些数据表明，STIM 1经典EF-手基序倾向于二聚化的功能在解决方案中，是负责传感变化[Ca 2 +]ER。
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.
登录
查看更多内容
DOI：
10.1002/prot.10080
发表时间：
2002-05-15
期刊：
PROTEINS-STRUCTURE FUNCTION AND GENETICS
影响因子：
--
作者：
Julenius, K;Robblee, J;Linse, S
通讯作者：
Linse, S
影响因子：
5.7
作者：
BODIAN, DL;JONES, EY;DAVIS, SJ
通讯作者：
DAVIS, SJ
DOI：
10.1073/pnas.0611713104
发表时间：
2007-03-06
影响因子：
11.1
作者：
Hauser, Christina T.;Tsien, Roger Y.
通讯作者：
Tsien, Roger Y.
影响因子：
2
作者：
Franchini, PLA;Reid, RE
通讯作者：
Reid, RE
影响因子：
15
作者：
Ye, YM;Lee, HW;Yang, JJ
通讯作者：
Yang, JJ