CysZ proteins_A family of sulfate transporters with remarkable architecture

说明(申请人提供)：硫是细胞生存所必需的元素，其在环境中最容易被利用的形式是硫酸盐。有三个已知的硫酸盐转运体家族负责细胞内对该离子的摄取。一个家族属于ABC转运蛋白家族，被认为在细胞外硫酸盐浓度较低且该过程发生所需能量时起作用。第二个属于主要促进剂超家族转运蛋白的SulP类。第三个家族是细菌CysZ基因家族的产物。当环境中的硫酸盐浓度高到足以使其通过化学渗透驱动机制进入细胞内时，SULP和CysZ被认为是活性的。我们测定了革兰氏阴性深海细菌Idiomarina loihiensis的CysZ的晶体结构至2.1°分辨率，并建立了该蛋白的功能分析方法。据我们所知，这是第一次报道原子分辨率的硫酸盐运输机的快照。这种结构显示了一种意想不到的新颖拓扑结构，其中蛋白质在膜平面上以紧密对称的二聚体的形式组装，因此分子的胞外和胞内两侧是相同的。两个二聚体沿着在膜中突出并平行于膜的疏水螺旋相互堆积，形成四聚体组装。该结构显示了结合的硫酸盐，但运输机制尚未揭开。我们计划进行旨在验证和理解CysZ非凡架构的实验(目标1)。我们还计划了解硫酸盐是如何通过膜运输的(目标2)，并表征触发CysZ离子电导孔打开的因素(目标3)。

DESCRIPTION (provided by applicant): Sulfur is an essential element for cell survival, and its most readily available form in the environment is sulfate. There are three known families of sulfate transporters that are responsible for intracellular uptake of this ion. One family belongs o the ABC transporter family and is thought to function when the concentration of extracellular sulfate is low and energy is required for the process to occur. A second one belongs to the SulP class of major facilitator superfamily transporters. A third family are the products of the bacteril CysZ gene family. SulPs and CysZ are thought to be active when the concentration of sulfate in the environment is high enough to allow its entry inside the cell by a chemiosmotic driven mechanism. We have determined the crystal structure to 2.1¿ resolution of CysZ from Idiomarina loihiensis, a gram- negative deep-sea water bacterium and have established a functional assay for this protein. To the best of our knowledge this represents the first report ofa snapshot of a sulfate transporter at atomic resolution. The structure shows an unexpected and novel topology in which the protein assembles as a tight symmetric dimer across the plane of the membrane, so that the extracellular and intracellular sides of the molecule are the same. Two dimers pack against each other along hydrophobic helices protruding in and parallel to the membrane, to form a tetrameric assembly. The structure shows bound sulfate but the mechanism of transport has yet to be unveiled. We plan to perform experiments aimed at validating and understanding the remarkable architecture of CysZ (Aim 1). We also plan to understand how sulfate is transported across the membrane (Aim 2), and to characterize the factors that trigger an opening of the CysZ ion conductance pore (Aim 3).