Protein Interactions in the Utilization of Iron by Bacteria

智力价值：铁是基本细胞过程中必需的营养物质，从氧气储存和运输到呼吸和DNA修复。病原菌具有铁摄取机制，使它们能够利用被乳铁蛋白、转铁蛋白或与血红素结合的宿主铁，从而克服其宿主生物体中遇到的低铁浓度。铁一旦到达细菌胞质中，人们对它的去向知之甚少。铁蛋白分子是细胞内铁浓度的动态调节者，但尽管它们在铁稳态中很重要，但人们对运送Fe2+储存的过程或促使其释放以安全整合新陈代谢的信号知之甚少。铜绿假单胞菌中有两个类似铁蛋白的分子：细菌铁蛋白(BFRB)，它由24个相同的亚基组成，与12个血红素分子结合；细菌铁蛋白(FtnA)，也由24个相同的亚基组装，但不结合血红素。最近发现，从BFRB释放铁需要电子由铁氧还蛋白(BFD)和铁氧还蛋白还原酶(FPR)介导，而从FtnA释放铁需要电子只由FPR介导。这些发现为研究蛋白质结构和蛋白质-蛋白质相互作用如何有助于病原菌中胞质铁的调节提供了一个独特的平台。为了研究这些蛋白质之间的相互作用，将采用包括生物化学、核磁共振光谱、X射线结晶学和计算生物学在内的多学科方法。具体目标是：1)研究并从结构上确定调节铁从BFRB和FtnA内腔流出的蛋白质-蛋白质相互作用。(2)挑战一种假设，即Fe2+通过在BFRB和FtnA的结构中的4个亚基的交叉处形成的四重气孔离开BFRB和FtnA的内部空洞。更广泛的影响：该项目的多学科性质将为所有学术水平的学生提供许多机会。通过每月的联合小组会议整合Rivera、Vakser和Lovell实验室(均位于堪萨斯大学)的努力，将使学生接触到培养多学科研究所必需的智力多样化的氛围。这一经历，再加上三个实验室(亚洲人、高加索人、西班牙人；男女比例约为40/60)存在的丰富的种族环境，将使研究生为毕业后可能遇到的多方面工作环境做好准备。同样重要的是，这个项目的协作精神将向学生展示基础广泛的方法在以下方面的好处：(1)解决复杂的问题；(2)更有效地将在一个实验室中获得的结果放在适当的背景下；从而(3)改善研究将影响更广泛的科学界，并最终造福于普通公众的前景。该项目由分子和细胞生物科学部的生物分子动力学、结构和功能簇以及化学部的生命过程化学计划共同支持。

Intellectual Merit: Iron is a required nutrient involved in fundamental cellular processes, from oxygen storage and transport to respiration and DNA repair. Pathogenic bacteria are equipped with iron uptake mechanisms that enable them to utilize host-iron sequestered by lactoferrin, transferrin or bound to heme, and therefore overcome the low-iron concentrations encountered in their host organisms. Much less is known about the fate of iron once it reaches the bacterial cytosol. Ferritin molecules function as dynamic regulators of cytosolic iron concentrations, but despite their importance in iron homeostasis, little is known about the processes that deliver Fe2+ for storage or the signals that prompt its release for safe integration in metabolism. There are two ferritin-like molecules in P. aeruginosa: a bacterioferritin (BfrB), which assembles from 24 identical subunits and binds 12 heme molecules, and a bacterial ferritin (FtnA), which is also assembled from 24 identical subunits but does not bind heme. It has recently been discovered that release of iron from BfrB requires electrons to be mediated by a ferredoxin (Bfd) and a ferredoxin reductase (FPR), whereas release of iron from FtnA requires that electrons be mediated only by FPR. These findings provide a unique platform to study how protein structure and protein-protein interactions contribute to the regulation of cytosolic iron in pathogenic bacteria. In order to investigate these protein-protein interactions a multidisciplinary approach will be undertaken which includes biochemistry, NMR spectroscopy, X-ray crystallography and computational biology. The specific goals are: 1) Investigate and structurally determine the protein-protein interactions that regulate the outflow of iron from the interior cavities of BfrB and FtnA. (2) Challenge a hypothesis that Fe2+ exits the BfrB and FtnA interior cavities via the four-fold pores formed in their structures at the intersection of 4-subunits.Broader Impacts: The multidisciplinary nature of the project will provide many opportunities for students at all academic levels. The integration of efforts in the Rivera, Vakser and Lovell laboratories (all located at the University of Kansas) via monthly joint group meetings will expose the students to the intellectually diverse atmosphere that is necessary to nurture multidisciplinary research. This experience, coupled to the ethnically rich environment present in the three labs (Asian, Caucasian, Hispanic; ~40/60 female/male) will prepare the graduate students well for the multifaceted work environment they are likely to encounter after graduation. Just as important, the collaborative spirit of this project will illustrate to students the benefits of a broad-based approach for (1) solving complex problems; (2) more effectively putting the results obtained in one laboratory into its proper context; and thus (3) improving the prospect that the research will impact the greater scientific community, and ultimately benefit the general public.This project is jointly supported by the Biomolecular Dynamics, Structure and Function Cluster in the Division of Molecular and Cellular Biosciences and the Chemistry of Life Processes program in the Chemistry Division.