Tracking Metal Flux Through a Pathogenic Export Complex

铜稳态失调对人类健康有多方面的影响。 哺乳动物细胞使用铜泵维持动态平衡，金属酸铜依赖 酶，将铜从一个细胞器/细胞/组织转移到另一个细胞器/细胞/组织。同样重要的是 细菌转运蛋白及其作为致病因子在病原体中的作用的研究。一个重要的 一类外排泵，存在于许多革兰氏阴性病原体中，是RND- 以大肠杆菌CusCBAF系统为例的典型重金属输出子 具有周质空间，对铜(I)和银(I)的输出具有选择性。这个建筑群是 由三种蛋白质组成，CusA是跨膜泵，CusB是一种可溶性的“接头”蛋白质， CusC是一种外膜孔。此外，出口机械要求有 CusF是一种小的可溶性周质伴侣，它将铜或银穿梭到三方复合体中。 尽管近年来取得了许多进展，但拼图中的关键一块一直缺失-- 金属转移的速度，以及该速度如何由特定的结构元素控制 相互作用的蛋白质成分。PI的实验室已经开发出一套动力工具 涉及硒蛋氨酸(SEM)替代与快速冷冻淬火(RFQ)混合和 XAS检测具有广泛的适用性。本提案旨在将这些工具应用于研究 CusCBAF系统的金属出口机理的分子细节。一共有三个 明确的目标。目标1将探索CusF独特的配位化学，其中包括一个 史无前例的色氨酸配体(W44)，覆盖该位点并产生荧光 发射波长为490 nm。CO与W44A变异体结合的新发现将用于测试 该假说认为W44的作用是保护该部位免受氧的氧化。AIM 2将使用 RFQ混合与XAS联用检测Se边Se-Cu/Ag跟踪金属动力学 从CusF转移到CusB，从CusF转移到CUSA，目标是测定残留量 这对金属出口的效率很重要。通过计算预测的转移络合物 蛋白质-蛋白质相互作用的研究将被隔离和表征。AIM 3将调查 组氨酸激酶的周质结构域对金属的感知机制 通过自动磷酸化和磷酸转移激活CUSR转录因子，测试 感觉域使用高亲和力和低亲和力金属结合位点来开启的假说 并上调其激酶活性。预期的结果是对 金属出口机制的分子细节是制定战略的前提 用于阻断致病金属抗性途径，降低毒力。

Dysregulation of copper homeostasis has multiple consequences for human health. Mammalian cells use copper pumps to maintain homeostasis, metalate copper-dependent enzymes, and transfer copper from one organelle/cell/tissue to another. Of equal significance is the study of bacterial transporters and their role as virulence factors in pathogens. An important class of efflux pump, present in a number of gram-negative pathogenic organisms is the RND- type heavy metal exporter exemplified by the CusCBAF system of E. coli which spans the periplasmic space and exhibits selectivity for export of Cu(I) and Ag(I). The complex is comprised of three proteins, CusA a transmembrane pump, CusB is a soluble “adaptor” protein, and CusC, an outer-membrane pore. In addition, the export machinery requires the presence of CusF, a small soluble periplasmic chaperone which shuttles Cu or Ag to the tripartite complex. Despite many advances in recent years, a critical piece of the puzzle has been missing – the rate at which metals transfer, and how that rate is controlled by specific structural elements of the interacting protein components. The PI’s laboratory has developed a suite of kinetic tools involving selenomethionine (SeM) substitution coupled to rapid freeze quench (RFQ) mixing and XAS detection which are broadly applicable. This proposal sets out to apply these tools to study the mechanisms of metal export by the CusCBAF system in molecular detail. There are three specific aims. Aim 1 will explore the unique coordination chemistry of CusF, which includes an unprecedented tryptophan ligand (W44) that caps the site and gives rise to fluorescence emission at 490 nm. The novel finding that CO binds to the W44A variant will be used to test the hypothesis that the role of W44 is to protect the site from oxidation via O2. Aim 2 will use RFQ mixing coupled to XAS detection of Se-Cu/Ag at the Se edge to follow the kinetics of metal transfer from CusF to CusB and from CusF to CusA with the goal of determining residues important for the efficiency of metal export. Transfer complexes predicted from computational studies of protein-protein interactions will be isolated and characterized. Aim 3 will investigate the mechanism of metal sensing by the periplasmic domain of the histidine kinase CusS which activates the CusR transcription factor via autophosphorylation and phosphotransfer, testing the hypothesis that the sensory domain uses high and low-affinity metal binding sites to switch on and upregulate its kinase activity. The expected outcome is an understanding of the mechanism of metal export in molecular detail, a prerequisite for the development of strategies for interrupting pathogenic metal resistance pathways, and diminishing virulence.