Ca2+ Binding Alters the Interdomain Flexibility between the Two Cytoplasmic Calcium-binding Domains in the Na+/Ca2+ Exchanger
Ca2+ Binding Alters the Interdomain Flexibility between the Two Cytoplasmic Calcium-binding Domains in the Na+/Ca2+ Exchanger
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DOI：
10.1074/jbc.m111.249268
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发表时间：
2011-09-16
影响因子：
4.8
通讯作者：
Brueschweiler, Rafael
中科院分区：
文献类型：
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作者：
Salinas, Roberto K.;Bruschweiler-Li, Lei;Brueschweiler, Rafael
Na+/Ca~(2+)交换器(NCX)是一种膜蛋白，它催化Na~+和Ca~(2+)离子跨质膜反向转运，在维持各种细胞内的钙稳态中起着关键作用。NCX由一个跨膜部分和一个大的细胞内环组成。NCX转运功能的激活需要钙离子与两个串联的C2结构域CBD1和CBD2结合，这两个结构域是交换器细胞内环的组成部分。虽然已经有了CBD1和CBD2的高分辨结构，但它们的结构域结构和动力学以及变构钙调节的原子水平机制仍不清楚。在这里，我们使用溶液核磁共振光谱来研究CBD12的域间动力学，CBD12是一个32 kDa的结构，包含CBD1和CBD2结构域，通过一个短连接子连接。对核磁共振残馀偶极耦合的分析表明，CBD12在无钙和有钙离子存在的情况下平均呈拉长形状。APO态的核磁共振N-15驰豫数据表明，这两个结构域在纳秒时间尺度上有广泛的相对排列。这些排列包括显著的非线性域间取向。钙离子与CBD1的结合显著限制了结构域间的灵活性，稳定了更刚性的拉长构象。这些发现提示了CBD12在NCX功能中的作用的分子机制。
The Na+/Ca2+ exchanger (NCX) is a membrane protein, which catalyzes the counter transport of Na+ and Ca2+ ions across the plasma membrane, playing a key role in the maintenance of the intracellular Ca2+ homeostasis in various cell types. NCX consists of a transmembrane part and a large intracellular loop. The activation of the NCX transport function requires the binding of Ca2+ to two tandem C2 domains, CBD1 and CBD2, which are an integral part of the exchanger's intracellular loop. Although high-resolution structures of individual CBD1 and CBD2 are available, their interdomain structure and dynamics and the atomic level mechanism of allosteric Ca2+-regulation remains unknown. Here, we use solution NMR spectroscopy to study the interdomain dynamics of CBD12, a 32 kDa construct that contains both the CBD1 and CBD2 domains connected by a short linker. Analysis of NMR residual dipolar couplings shows that CBD12 assumes on average an elongated shape both in the absence and in the presence of Ca2+. NMR N-15 relaxation data of the Apo state indicate that the two domains sample a wide range of relative arrangements on the nanosecond time scale. These arrangements comprise significantly non-linear interdomain orientations. Binding of Ca2+ to CBD1 significantly restricts the interdomain flexibility, stabilizing a more rigid elongated conformation. These findings suggest a molecular mechanism for the role of CBD12 in the function of NCX.