PDF(Pubmed)
Abstract：
Sodium-calcium exchanger (NCX) proteins are ubiquitously expressed and play a pivotal role in cellular calcium homeostasis by mediating uphill calcium efflux across the cell membrane. Intracellular calcium allosterically regulates the exchange activity by binding to two cytoplasmic calcium-binding domains, CBD1 and CBD2. However, the calcium-binding affinities of these domains are seemingly inadequate to sense physiological calcium oscillations. Previously, magnesium binding to either domain was shown to tune their affinity for calcium, bringing it into the physiological range. However, while the magnesium-binding site of CBD2 was identified, the identity of the CBD1 magnesium site remains elusive. Here, using molecular dynamics in combination with differential scanning fluorimetry and mutational analysis, we pinpoint the magnesium-binding site in CBD1. Specifically, among four calcium-binding sites (Ca1-Ca4) in this domain, only Ca1 can accommodate magnesium with an affinity similar to its free intracellular concentration. Moreover, our results provide mechanistic insights into the modulation of the regulatory calcium affinity by magnesium, which allows an adequate NCX activity level throughout varying physiological needs.
摘要：
钠钙交换蛋白（NCX）广泛表达，并通过介导跨细胞膜的上坡钙流出在细胞钙稳态中起关键作用。细胞内钙通过与两个细胞质钙结合结构域结合来变构调节交换活性,CBD1和CBD2。然而,这些结构域的钙结合亲和力似乎不足以感知生理钙振荡。以前,镁与任一结构域的结合被证明可以调节它们对钙的亲和力，把它带到生理范围内。然而,同时鉴定了CBD2的镁结合位点，CBD1镁位点的身份仍然难以捉摸。这里,使用分子动力学结合差示扫描荧光分析法和突变分析，我们确定了CBD1中的镁结合位点。具体来说,在该结构域的四个钙结合位点（Ca1-Ca4）中，只有Ca1可以以与其游离细胞内浓度相似的亲和力容纳镁。此外，我们的结果提供了对镁调节钙亲和力的机制见解，这允许在整个不同的生理需求中具有足够的NCX活性水平。
