Structure and Function of Bacterial CNNM Magnesium Transporters

细菌CNNM镁转运蛋白的结构和功能

• 批准号: RGPIN-2020-07195
• 负责人: Gehring, Kalle
• 金额: $ 3.29万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744076
• 关键词: Structure; Function; Bacterial; CNNM; Magnesium

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.

镁 (Mg2+) 是细胞内最丰富的二价阳离子，对于多种生化活动至关重要。尽管细胞膜上的 Mg2+ 梯度并不大，但越来越多的证据表明，细胞内 Mg2+ 的变化通过许多酶和代谢传感器对 Mg2+ 的需求来调节细胞生长。细胞 Mg2+ 稳态的重要性体现在细菌和真核生物中大量的 Mg2+ 转运蛋白。在人类中，镁吸收或沉积的遗传性疾病是由瞬时受体电位褪黑素 6 型 (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 的突变引起的。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的机制。 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+ 稳态中发挥重要作用。 CNNM 家族由保守的跨膜结构域（DUF21 - 未知功能结构域 21）和胞质胱硫醚 β-合酶 (CBS) 对结构域定义。虽然与 Mg2+ 转运相关，但尚不清楚 CNNM 蛋白本身是否是 Mg2+ 转运蛋白，或者它们是否调节其他蛋白质的转运。在人类中，CNNM 蛋白的突变会导致多种与 Mg2+ 和 Ca2+ 吸收或沉积相关的疾病。在低等真核生物和细菌中，CNNM 蛋白的突变会导致对金属毒性的抵抗和 Mg2+ 运输的缺陷。我们的研究小组已经确定了CNNM胞质结构域的多种结构，并表明它们在与Mg2+·ATP结合后会发生较大的构象变化。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有 DUF21 的蛋白质。多种蛋白质在多种条件下都能良好表达和结晶。我们还使用负染色电子显微镜 (EM) 研究一种细菌蛋白质。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 结构域是功能未知的最大结构域家族，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 个 CNNM 直向同源物，但我们尚不了解它们精确的生化功能。所提出的工作将加深我们对 CNNM 蛋白在二价阳离子运输中的功能及其通过 Mg2+·ATP 结合的调节的理解。