Structure and Function of IscA, a Potential Iron Delivery Protein for Iron-Sulfur Cluster Assembly

铁硫簇是生物学中最普遍的氧化还原辅助因子之一。在整个进化过程中，铁硫团簇已成为能量转化、糖代谢、固氮、血红素和生物素生物合成、DNA合成和DNA修复以及基因表达调控等多种生理过程的组成部分。至少有六种蛋白质（IscS, IscU, IscA, HscB， HscA和铁氧还蛋白）由基因簇iscSUA-hscBA-fdx编码，已被确定为细菌中铁硫簇的一般生物发生所必需的。在真核生物中也发现了这6种蛋白的同源物，表明铁硫簇组装机制是高度保守的。研究表明，半胱氨酸脱硫酶IscS为铁硫簇提供硫，IscU是IscS介导的铁硫簇组装的支架，并最终将铁硫簇转移到靶蛋白上。然而，铁硫团簇的铁供体在很大程度上仍然是难以捉摸的。本项目旨在验证一个假设，即IscA的主要功能是招募细胞内铁并为IscU中铁硫簇的组装提供铁，并且IscA介导的铁向IscU的转移是通过蛋白质-蛋白质相互作用协调的。本研究有两个具体目的：1)结合生物化学、生物物理和x射线晶体学等方法，确定IscA的铁结合特性，确定对铁结合有重要作用的氨基酸残基，确定铁中心的氧化还原特性。2)通过探究IscA与IscU和IscS的蛋白-蛋白相互作用，探讨铁从IscA向IscU中铁硫簇转移的机制。这一结果将为铁硫簇的一般生物发生提供基础知识，这一过程从细菌到人类都是保守的。此外，了解铁硫团簇生物发生的铁传递将提供细胞内铁如何在其他生物过程中被利用的分子细节。更广泛的影响：该项目预计将吸引和留住高素质的研究生和本科生。学生不仅将学习多学科的方法来进行拟议的研究，而且还将通过积极的研究活动培养对他们未来在学术界或工业界的职业生涯至关重要的态度。将作出特别努力，招募积极性很高的少数民族和妇女研究生加入研究小组。这些研究项目将进一步整合到PI和Co-PI在其所在机构教授的生物化学课程中，并将极大地促进该机构生物化学课程的发展。

Iron-sulfur clusters are one of the most ubiquitous redox co-factors in biology. Throughout evolution, iron-sulfur clusters have become integral part of diverse physiological processes such as energy conversion, sugar metabolism, nitrogen fixation, heme and biotin biosynthesis, DNA synthesis and DNA repair, and regulation of gene expression. At least six proteins (IscS, IscU, IscA, HscB, HscA and Ferredoxin) encoded by a gene cluster iscSUA-hscBA-fdx have been identified as essential for the general biogenesis of iron-sulfur clusters in bacteria. The homologs of the six proteins have also been found in eukaryotic organisms, indicating that the iron-sulfur cluster assembly mechanism is highly conserved. It has been shown that IscS, a cysteine desulfurase, provides sulfur for iron-sulfur clusters, and that IscU is a scaffold for the IscS-mediated assembly of iron-sulfur clusters and eventually transfers iron-sulfur clusters to target proteins. However, the iron donor for iron-sulfur clusters largely remains elusive. This project aims to test a hypothesis that the primary function of IscA is to recruit intracellular iron and deliver iron for the assembly of iron-sulfur clusters in IscU, and that the IscA-mediated iron transfer to IscU is coordinated through the protein-protein interactions. The research has two specific aims: 1) To define the iron binding properties of IscA by combining the biochemical, biophysical and X-ray crystallographic approaches to identify the amino acid residues that are important for the iron binding and to determine the redox properties of the iron center. 2) To investigate the mechanism of iron transfer from IscA to the iron-sulfur clusters in IscU by exploring the protein-protein interactions of IscA with IscU and IscS. The results will provide fundamental knowledge on the general biogenesis of iron-sulfur clusters, a process that is conserved from bacteria to humans. In addition, understanding iron delivery for the biogenesis of iron-sulfur clusters will offer the molecular details on how intracellular iron may be utilized in other biological processes.Broader Impacts: The project is expected to attract and retain high quality graduate and undergraduate students. The students will not only learn the multidisciplinary methodology to conduct the proposed research, but also develop the attitude crucial to their future career in academia or industries through vigorous research activities. Special efforts will be taken to recruit highly motivated minority and woman graduate students to the research group. The research projects will further be integrated into the biochemistry courses that the PI and Co-PI have been teaching at their institution, and should greatly benefit the institutional biochemistry curricula.