Crystal Structure of Calmodulin Binding Domain of Orai1 in Complex with Ca2+•Calmodulin Displays a Unique Binding Mode
Crystal Structure of Calmodulin Binding Domain of Orai1 in Complex with Ca2+•Calmodulin Displays a Unique Binding Mode
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DOI：
10.1074/jbc.m112.380964
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发表时间：
2012-12-14
影响因子：
4.8
通讯作者：
Birnbaumer, Lutz
中科院分区：
文献类型：
--
作者：
Liu, Yanshun;Zheng, Xunhai;Birnbaumer, Lutz
Orai1是一种四聚体形式的质膜蛋白，通过与Ca2+耗尽传感器STIM1的相互作用，负责钙从细胞外环境流入胞质溶胶。随后是快速Ca2+中心点钙调蛋白（CaM）依赖性抑制，由CaM结合到称为钙调蛋白结合域（CMBD）的Orai1区域引起。Orai1和CaM在原子水平上的相互作用尚不清楚。在这里，我们报道了CaM中心点Orai1-CMBD复合物的晶体结构，显示一个CMBD与CaM的c端叶结合，不同于其他CaM靶蛋白复合物，CaM的N和c端叶（CaM-N和CaM- c）都参与靶结合。Orai1-CMBD主要通过疏水相互作用与CaM-C结合，主要涉及Orai1-CMBD残基Trp（76）与CaM-C疏水口袋相互作用。然而，核磁共振数据、等温滴定量热数据和下拉实验表明，CaM-N和CaM-C都可以结合Orai1-CMBD，但CaM-N的亲和力比CaM-C弱4倍。Orai1-CMBD（W76E）突变体的下拉实验、凝胶过滤色谱数据和NOE信号表明，CaM-N和CaM-C分别可以结合一个Orai1-CMBD。因此，我们的研究支持了一种不同寻常的、扩展的CaM与Orai1- cmbd的1:1结合模式，并量化了Orai1对CaM的亲和力。我们提出了CaM依赖的Orai1失活的两步机制，首先将CaM的C-lobe结合到一个Orai1的CMBD上，然后将CaM的N-lobe结合到邻近的Orai1的CMBD上。
Orai1 is a plasma membrane protein that in its tetrameric form is responsible for calcium influx from the extracellular environment into the cytosol in response to interaction with the Ca2+-depletion sensor STIM1. This is followed by a fast Ca2+center dot calmodulin (CaM)-dependent inhibition, resulting from CaM binding to an Orai1 region called the calmodulin binding domain (CMBD). The interaction between Orai1 and CaM at the atomic level remains unknown. Here, we report the crystal structure of a CaM center dot Orai1-CMBD complex showing one CMBD bound to the C-terminal lobe of CaM, differing from other CaM-target protein complexes, in which both N- and C-terminal lobes of CaM (CaM-N and CaM-C) are involved in target binding. Orai1-CMBD binds CaM-C mainly through hydrophobic interactions, primarily involving residue Trp(76) of Orai1-CMBD, which interacts with the hydrophobic pocket of CaM-C. However, NMRdata, isothermal titration calorimetry data, and pull-down assays indicated that CaM-N and CaM-C both can bind Orai1-CMBD, with CaM-N having similar to 4 times weaker affinity than CaM-C. Pulldown assays of a Orai1-CMBD(W76E) mutant, gel filtration chromatography data, and NOE signals indicated that CaM-N and CaM-C can each bind one Orai1-CMBD. Thus our studies support an unusual, extended 1: 2 binding mode of CaM to Orai1-CMBDs, and quantify the affinity of Orai1 for CaM. We propose a two-step mechanism for CaM-dependent Orai1 inactivation initiated by binding of the C-lobe of CaM to the CMBD of one Orai1 followed by the binding of the N-lobe of CaM to the CMBD of a neighboring Orai1.