Magnesium Transporter MgtA revealed as a Dimeric P-type ATPase

Magnesium (Mg2+) uptake systems are present in all domains of life given the vital role of this ion. Bacteria acquire Mg2+ via conserved Mg2+ channels and transporters. The transporters are required for growth when Mg2+ is limiting or during bacterial pathogenesis, but, despite their significance, there are currently no known structures for these transporters. Here we report the first structure of the Mg2+ transporter MgtA solved by single particle cryo-electron microscopy (cryo-EM). We obtained high resolution structures of both a homodimeric form (2.9 A), the first for a P-type ATPase, and a monomeric form (3.6 A). Each monomer unit of MgtA displays a structural architecture that is similar to other P-type ATPases with a transmembrane domain and two soluble domains. The dimer interface consists of contacts between residues in adjacent soluble nucleotide binding and phosphotransfer regions of the haloacid dehalogenase (HAD) domain. The ATP binding site and consequences of nucleotide binding were characterized by a combination of cryo-EM, molecular dynamics simulations, hydrogen-deuterium exchange mass spectrometry, and mutagenesis. Finally, our structure revealed a Mg2+ ion in the transmembrane segments, which, when combined with sequence conservation and mutagenesis studies, allowed us to propose a model for Mg2+ transport across the lipid bilayer. Our dimeric structures of MgtA also reveal regulatory features that have implications for related P-type ATPases such as the calcium pump SERCA.

鉴于镁离子（Mg²+）的关键生理功能，所有生命域中均存在其摄取系统。细菌通过保守的镁离子通道与转运蛋白获取Mg²+。当镁离子匮乏或细菌致病时，这类转运蛋白是细菌生长所必需的；尽管其功能意义重大，但目前尚无这类转运蛋白的已知三维结构。本研究首次通过单颗粒冷冻电子显微镜（cryo-electron microscopy, cryo-EM）解析了镁离子转运蛋白MgtA的结构。我们获得了同二聚体形式（分辨率2.9埃，为P型ATP酶（P-type ATPase）的首个此类结构）以及单体形式（分辨率3.6埃）的高分辨率结构。MgtA的每个单体亚基的结构架构与其他P型ATP酶类似，包含一个跨膜结构域与两个可溶性结构域。二聚体界面由相邻单体的可溶性核苷酸结合区域与卤酸脱卤酶（haloacid dehalogenase, HAD）结构域的磷酸转移区域的残基相互作用构成。我们结合冷冻电镜、分子动力学模拟、氢氘交换质谱与诱变实验，对ATP结合位点以及核苷酸结合的效应进行了表征。最后，我们的结构在跨膜区段中发现了一个镁离子，结合序列保守性与诱变实验结果，我们得以提出镁离子跨脂质双层转运的模型。我们解析的MgtA同二聚体结构还揭示了调控特征，这对钙泵SERCA等相关P型ATP酶的研究具有参考价值。