Structure and Function of Bacterial CNNM Magnesium Transporters

镁是细胞内含量最丰富的二价阳离子，是多种生化活动所必需的。虽然在细胞膜上没有很大的镁梯度，但越来越多的证据表明，细胞内镁的变化是通过许多酶和代谢感受器对镁的需求来调节细胞生长的。细菌和真核生物中大量的镁转运体都体现了细胞内镁的动态平衡的重要性。在人类中，镁摄取或沉积的遗传性疾病由瞬时受体潜力蛋白6(TRPM6)、镁转运体1(MagT1)、溶质载体家族41成员(Slc41a1-3)、claudin 16和CNNms突变引起。在细菌中，镁转运体MgtE和Cora的晶体结构揭示了离子选择性和门控的机制。CNNM蛋白在真核生物和原核生物中都是保守的，在mG2的动态平衡中起着重要的作用，因此引起了人们的特别关注。CNNM家族是由一个保守的跨膜结构域(DUF21--未知功能的结构域)和胞浆中的胱硫醚-β-合成酶(CBS)对结构域定义的。虽然与mg2转运有关，但cnnm蛋白本身是mg2转运蛋白还是通过其他蛋白质调节转运尚不清楚。在人类中，CNNM蛋白的突变会导致与镁和钙摄取或沉积相关的各种疾病。在低等真核生物和细菌中，CNNM蛋白的突变导致对金属毒性的抗性和镁离子转运的缺陷。本课题组已经确定了CNNM胞浆结构域的多种结构，并表明它们在与Mg2·ATP结合时发生了很大的构象变化。在这里，我们建议确定细菌CNNM蛋白功能和调控的结构基础。我们有两个目标：1.结构研究。我们已经克隆并表达了来自20多个物种的含有DUF21-的蛋白。几种蛋白质在多种条件下都能很好地表达和结晶。我们还用负染色电子显微镜(EM)研究了一种细菌蛋白。我们将通过EM或X射线结晶学来确定CNNM细菌同源基因的结构。2.功能研究。我们将使用生物物理方法和功能分析来表征CNNM蛋白的功能和构象变化。这些研究将解决DUF21结构域是否直接或间接调节离子转运的问题，并揭示它们的胞浆结构域如何调节它们的功能。CNNM蛋白的DUF21结构域是最大的未知功能结构域家族，目前还没有已知的结构。在植物、动物和细菌中已知的CNNM同源基因有5万多个，但我们还不清楚它们的确切生化功能。这些工作将有助于我们进一步了解CNNM蛋白在二价阳离子转运中的作用，以及它们受Mg2·ATP结合的调控。

Magnesium (Mg2+) is the most abundant divalent cation inside cells and essential for a wide variety of biochemical activities. Although there is not a large gradient of Mg2+ across the cell membrane, increasing evidence suggests that changes in intracellular Mg2+ modulate cell growth through the requirement of many enzymes and metabolic sensors for Mg2+. The importance of cellular Mg2+ homeostasis is manifest in the large number of Mg2+ transporters both in bacteria and eukaryotes. In humans, hereditary diseases of magnesium uptake or deposition arise from mutations in transient receptor potential melastatin type 6 (TRPM6), Mg2+ transporter 1 (MagT1), solute carrier family 41 members (Slc41a1-3), claudin 16, and CNNMs. In bacteria, crystal structures for the Mg2+ transporters MgtE and CorA have revealed the mechanisms for ion selectivity and gating. The CNNM proteins are of particular interest as they are conserved across both eukaryotes and prokaryotes and play essential roles in Mg2+ homeostasis. The CNNM family is defined by a conserved transmembrane domain (DUF21 -- domain of unknown function 21) and a cytosolic cystathionine beta--synthase (CBS) pair domain. While associated with Mg2+ transport, it is unknown if CNNM proteins are themselves Mg2+ transporters or if they regulate transport by other proteins. In humans, mutations in CNNM proteins cause a variety of diseases related to Mg2+ and Ca2+ uptake or deposition. In lower eukaryotes and bacteria, mutations in CNNM proteins lead to resistance to metal toxicity and defects in Mg2+ transport. Our group has determined multiple structures of CNNM cytosolic domains and shown that they undergo a large conformational change upon Mg2+·ATP binding. Here, we propose to determine the structural basis of bacterial CNNM protein function and regulation. We have two aims: 1. Structural studies. We have cloned and expressed DUF21--containing proteins from over 20 species. Several proteins are well expressed and crystallize under multiple conditions. We have also used negative stain electron microscopy (EM) to study one bacterial protein. We will determine the structure of a CNNM bacterial ortholog by either EM or X--ray crystallography. 2. Functional studies. We will characterize the function and conformational changes of CNNM proteins using biophysical methods and functional assays. These studies will address the question of whether DUF21 domains directly or indirectly mediate ion transport and reveal how their cytosolic domains regulate their function. The DUF21 domain of CNNM proteins is the largest family of domains of unknown function and there are no known structures. There are well over 50,000 CNNM orthologs known in plants, animals and bacteria yet we do not understand their precise biochemical function. The work proposed will advance our understanding of the function of the CNNM proteins in divalent cation transport and their regulation by Mg2+·ATP binding.