Structure and Function of Bacterial CNNM Magnesium Transporters

镁（Mg2+）是细胞内最丰富的二价阳离子，对多种生化活动至关重要。尽管Mg2+在细胞膜上的梯度不大，但越来越多的证据表明，细胞内Mg2+的变化通过许多酶和Mg2+代谢传感器的需求来调节细胞生长。在细菌和真核生物中都存在大量的Mg 2+转运蛋白，这表明细胞内Mg 2+稳态的重要性。在人类中，镁摄取或沉积的遗传性疾病由瞬时受体电位6型黑素蛋白（TRPM6）、Mg2+转运蛋白1（MagT1）、溶质载体家族41成员（Slc41a1 - 3）、密蛋白16和CNNM的突变引起。在细菌中，Mg2+转运蛋白MgtE和CorA的晶体结构揭示了离子选择性和门控机制。CNNM蛋白是特别感兴趣的，因为它们在真核生物和原核生物中是保守的，并且在Mg 2+稳态中发挥重要作用。CNNM家族由保守的跨膜结构域（DUF21-未知功能的结构域21）和胞质胱硫醚β-合酶（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+转运蛋白或它们是否通过其他蛋白调节转运尚不清楚。在人类中，CNNM蛋白的突变导致与Mg2+和Ca2+摄取或沉积相关的多种疾病。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Mg 2+转运的缺陷。我们的小组已经确定了CNNM胞质结构域的多种结构，并表明它们在Mg 2+·ATP结合后发生了大的构象变化。在这里，我们建议确定细菌CNNM蛋白功能和调节的结构基础。我们有两个目标：1。结构研究。我们已经从20多个物种中克隆并表达了含DUF 21的蛋白。几种蛋白质在多种条件下充分表达并结晶。我们还使用负染电子显微镜（EM）研究了一种细菌蛋白。我们将通过EM或X射线晶体学确定CNNM细菌直系同源物的结构。2.功能研究。我们将使用生物物理学方法和功能测定来表征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介导离子转运的问题，并揭示其胞质结构域如何调节其功能。CNNM蛋白的DUF21结构域是最大的功能未知的结构域家族，并且没有已知的结构。在植物、动物和细菌中已知有超过50，000种CNNM直系同源物，但我们不了解它们的精确生化功能。这些工作将进一步加深我们对CNNM蛋白在二价阳离子转运中的功能及其与Mg~（2+）·ATP结合的调控的认识。

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.