Magnesium Homeostasis in Microorganisms

镁化学在生物阳离子中是独一无二的，细胞具有新的机制来 调节镁离子并促进其通过细胞膜。这项提案的重点是CORA 镁离子转运体，大多数真细菌和古生菌的主要镁离子吸收系统。水晶 来自Thermotoga Sea的CORA的结构已被确定为3.9A分辨率。这是一个 具有两个跨膜(TM)螺旋的漏斗形高五聚体。胞外区是 仅由连接两个TM螺旋的一个短的保守环组成。航道编排完成 五个螺旋，每个单体一个，看起来被巨大的疏水侧链控制 毛孔内的残留物。这些螺旋通过细胞质延伸到膜之外。 区域，形成漏斗内表面。在漏斗外面，毛孔的细胞质颈部是 被一环高度保守的带正电的残基包围。两个带负电荷的螺旋 在细胞质区域中向后延伸到漏斗外的膜并毗邻 带正电荷。这些外部螺旋可能用来抵消带正电的环，这表明 一种门控装置。一个明显的镁离子结合在单体之间的细胞质区域， 将延伸的螺旋从一个单体连接到另一个单体中的一组螺旋。镁离子结合部位， 在Cora同源基因中保守，可能将孔洞开放与细胞内镁离子浓度联系起来。 目标1将继续利用定点定向技术研究CORA及其镁离子转运机制 突变和运输试验，以探索运输和门控的假想机制。 AIM2将继续进行毛滴虫Cora的结构工作，包括确定一个开放的气孔 并对鼠伤寒沙门氏菌的可溶性区结构进行了测定。 Aim 3将专注于Cora大家庭的其他成员。细菌ZntB具有适度的序列 与CORA相同，但介导了锌离子的外流，而不是镁离子的内流。真核细胞MRS-2蛋白介导 Mg~(2+)进入线粒体，但与CORA仅在膜区有序列相似性。我们 提出了一种假设，即ZntB和Mr_2的结构与CoLa的Mg~(2+)相同 通过测定两个转运蛋白的可溶性域的晶体结构来确定转运蛋白。 与健康相关。CORA的研究很重要，因为它是原核生物中的一个毒力因子，因此是一种 抗生素的靶标，此外，它还介导镁离子进入线粒体，因此在控制 真核细胞中的镁离子动态平衡以及线粒体在细胞凋亡中的作用。

Mg chemistry is unique among biological cations, and cells possess novel mechanisms for regulating Mg2+ and facilitating its passage through membranes. This proposal focuses on the CorA Mg2+ transporter, the primary Mg2+ uptake system for most Eubacteria and Archaea. The crystal structure of CorA from Thermotoga maritime has been determined to 3.9 A resolution. It is a funnel-shaped homopentamer with two transmembrane (TM)helices. The extracellular region is composed of only a short conserved loop that connects the two TM helices. The channel is composed of five helices, one from each monomer and appears gated by the side chains of bulky hydrophobic residues within the pore. These helices extend well beyond the membrane through the cytoplasmic domain, forming the funnel inner surface. Outside the funnel, the cytoplasmic neck of the pore is surrounded by a ring of highly conserved positively charged residues. Two negatively charged helices in the cytoplasmic domain extend back towards the membrane outside the funnel and abut the ring of positive charge. These exterior helices may serve to counteract the positively charged ring, suggesting a gating mechanism. An apparent Mg2+ ion was bound in the cytoplasmic domain between monomers, linking the extended helix from one monomer to a set of helices in another. The Mg2+ binding site, conserved in CorA orthologs, may link pore opening to the intracellular concentration of Mg2+. Aim 1 will continue study of CorA and its mechanism of Mg2+ transport using site directed mutagenesis and transport assays to probe the hypothesized mechanism of transport and gating. Aim2 will continue structural work on the T. maritima CorA including determination of an open pore form of CorA and to determine the structure of the soluble domain of S. Typhimurium CorA. Aim 3 will focus on other members of the large CorA family. Bacterial ZntB has modest sequence identity to CorA but mediates efflux of Zn2+ rather than influx of Mg2+. Eukaryotic Mrs2 proteins mediate Mg2+ influx into mitochondria but have sequence similarity with CorA only in the membrane domain. We propose to test the hypothesis that the structures of ZntB and Mrs2 are identical to that of the CorA Mg2+ transporter by determining the crystal structures of the soluble domains of both transporters. Health relevance. Study of CorA is important because it is a virulence factor in prokaryotes and thus an antibiotic target, In addition, it mediate Mg2+ flux into mitochondria and thus is important in control of Mg2+ homeostasis in eukaryotic cells and possibly in the mitochondrion's role in apoptosis.