Protein Interactions in the Utilization of Iron by Bacteria

智力优势：铁是一种必需的营养物质，参与基本的细胞过程，从氧气储存和运输到呼吸和DNA修复。致病菌具有铁摄取机制，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，从而克服宿主体内的低铁浓度。对于铁到达细菌细胞质后的命运，我们所知甚少。铁蛋白分子作为胞内铁浓度的动态调节剂发挥作用，但尽管它们在铁稳态中很重要，但人们对铁离子储存的传递过程或促使其释放以安全整合代谢的信号知之甚少。铜绿假单胞菌中有两种类似铁蛋白的分子：一种是细菌铁蛋白（BfrB），由24个相同的亚基组装而成，并结合12个血红素分子；另一种是细菌铁蛋白（FtnA），也由24个相同的亚基组装而成，但不结合血红素。最近发现，从BfrB中释放铁需要由铁氧化还蛋白（Bfd）和铁氧化还蛋白还原酶（FPR）介导的电子，而从FtnA中释放铁只需要由FPR介导的电子。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促进致病菌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-蛋白质相互作用，将采取多学科方法，包括生物化学，核磁共振波谱学，x射线晶体学和计算生物学。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)挑战了一个假设，即Fe2+通过在BfrB和FtnA结构中4个亚基相交处形成的四重孔排出BfrB和FtnA内部腔。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。Rivera， Vakser和Lovell实验室（均位于堪萨斯大学）通过每月的联合小组会议将努力整合在一起，这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。这种经历，再加上三个实验室中种族丰富的环境（亚洲人、高加索人、西班牙人；男女比例约为40/60），将为研究生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，这个项目的合作精神将向学生们展示一个广泛的方法的好处：(1)解决复杂问题；(2)更有效地将一个实验室获得的结果置于适当的环境中；从而(3)提高研究将影响更大的科学界的前景，并最终使公众受益。本项目由分子与细胞生物科学部生物分子动力学、结构与功能研究小组和化学部生命过程化学研究小组共同支持。

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.