Structure and Function of Bacterial CNNM Magnesium Transporters

镁（Mg2+）是细胞内最丰富的二价阳离子，对多种生物化学活动至关重要。尽管Mg2+在细胞膜上没有很大的梯度，但越来越多的证据表明，细胞内Mg2+的变化通过许多酶和代谢传感器对Mg2+的需求来调节细胞生长。细胞内Mg2+稳态的重要性体现在细菌和真核生物中大量的Mg2+转运体中。在人类中，镁摄取或沉积的遗传性疾病是由瞬时受体电位美拉他汀6型（TRPM6）、Mg2+转运体1 （MagT1）、溶质载体家族41成员（Slc41a1-3）、claudin 16和CNNMs突变引起的。在细菌中，Mg2+转运体MgtE和CorA的晶体结构揭示了离子选择性和门控机制。CNNM蛋白特别令人感兴趣，因为它们在真核生物和原核生物中都是保守的，并且在Mg2+稳态中起重要作用。CNNM家族由一个保守的跨膜结构域（DUF21 -未知功能结构域21）和一个胞质胱硫氨酸-合成酶（CBS）对结构域定义。虽然与Mg2+转运有关，但CNNM蛋白本身是否是Mg2+转运体，或者它们是否通过其他蛋白质调节转运尚不清楚。在人类中，CNNM蛋白的突变导致多种与Mg2+和Ca2+摄取或沉积相关的疾病。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Mg2+运输缺陷。我们的团队已经确定了CNNM细胞质结构域的多个结构，并表明它们在Mg2+·ATP结合时发生了很大的构象变化。在此，我们提出确定细菌CNNM蛋白功能和调控的结构基础。我们有两个目标：1。结构的研究。我们已经克隆并表达了20多个物种的DUF21蛋白。几种蛋白质在多种条件下都能很好地表达和结晶。我们也用阴性染色电镜（EM）研究了一种细菌蛋白。我们将通过EM或X射线晶体学确定CNNM细菌同源物的结构。2. 功能的研究。我们将利用生物物理方法和功能分析表征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介导离子转运的问题，并揭示其细胞质结构域如何调节其功能。CNNM蛋白的DUF21结构域是最大的未知功能结构域家族，没有已知的结构域。在植物、动物和细菌中已知的CNNM同源物超过5万种，但我们还不清楚它们的确切生化功能。这项工作将促进我们对CNNM蛋白在二价阳离子运输中的功能及其通过Mg2+·ATP结合的调控的理解。

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.