CAREER: Elucidating the role of ATP in Cytosolic Iron Sulfur Cluster Biogenesis

波士顿大学的Deborah Perlstein教授获得了生命过程化学项目的这一职业奖，他正在研究铁和硫簇是如何组装起来的，并转移到真核细胞胞浆中相应的蛋白质中。簇的生物合成对细胞的生长和分裂是必不可少的，因为需要铁硫簇的蛋白质在DNA复制和修复以及许多其他重要的细胞功能中发挥着核心作用。尽管它的重要性毋庸置疑，但科学家们对这些铁硫簇是如何合成并最终转移到适当的蛋白质/酶的分子细节知之甚少。珀尔斯坦教授的实验室结合了遗传学、生物化学和生物物理学的方法，阐明了蛋白质支架上簇的组装过程，以及最终将簇插入蛋白质所需的后续步骤。这项研究为本科生和研究生以及博士后研究员提供了一个极好的培训环境，使他们具备在化学-生物界面成为研究领导者所需的专业知识。此外，通过制定与研究计划密切相关的本科生生物化学讲座和实验室课程的课程，以及K-12 STEM扩展计划，Perlstein教授的工作扩大了人们对生物化学和生物技术研究的兴趣，这些研究构成了波士顿地区就业机会的重要组成部分。该项目的具体目标是揭示为什么酵母胞质铁硫簇支架蛋白需要ATP水解才能发挥其活性。由于酵母酶及其在簇状支架ATPase的MRP/Nbp35家族中的同源物形成了异常Walker A家族的P-loop NTPase家族中的一个不同的亚家族，Perlstein博士和她的团队假设ATP水解被用来诱导支架内的构象变化，该构象变化被用来协调支架上新生的簇的组装和该簇向受体的转移。这种核苷酸驱动的开关可以通过簇支架位点和ATPase位点之间的直接变构通信，通过调节与其他簇生物发生蛋白的动态相互作用，或者通过这两种机制的组合来影响簇支架的组装和/或转移功能。通过确定ATPase突变体如何在体外影响簇支架活性，确定与支架相互作用的其他因素，以及阐明支架簇或其他簇组装因素如何影响ATP水解的动力学机制，研究人员有望揭示这一ATP水解铁硫簇支架大家族是如何利用核苷酸水解来协调支架内新生簇的组装，以及随后将簇从支架动员到靶点所需的运输步骤。这项研究与一项教育计划相结合，旨在通过与波士顿大学的学习资源网络的合作，使K-12学生在化学-生物界面上获得研究和职业机会。此外，珀尔斯坦教授领导为化学和生物专业的本科生开发了一门生物化学强化课程，以满足计划从事研究职业的学生的需求。

With this CAREER award from the Chemistry of Life Processes Program, Professor Deborah Perlstein at Boston University is investigating how clusters of iron and sulfur are assembled and transferred to their appropriate proteins in the cytosol of eukaryotic cells. Cluster biosynthesis is essential for cell growth and division since proteins requiring iron sulfur clusters play central roles in DNA replication and repair as well as many other important cellular functions. Despite its unquestionable importance, scientists understand remarkably little about the molecular details of how these iron sulfur clusters are synthesized and ultimately transferred into the appropriate proteins/enzymes. Professor Perlstein's lab is using a combination of genetic, biochemical, and biophysical approaches to shed light on how assembly of a cluster on a protein scaffold takes place as well as on the subsequent steps required to ultimately insert the cluster into a protein. The research provides an excellent training environment for students at the undergraduate and graduate students as well as postdoctoral fellows, equipping them with the expertise required to become leaders in research at the chemistry-biology interface. Furthermore, by developing the curriculum for an undergraduate biochemistry lecture and laboratory course and a K-12 STEM outreach program both intimately connected with the research plan, Professor Perlstein's work broadens interest in biochemistry and biotechnology research which forms a significant portion of the job opportunities in the Boston area. The specific goal of this project is to uncover why the yeast cytosolic iron sulfur cluster scaffolding proteins require ATP hydrolysis for their activity. Since the yeast enzyme and its homologs in the Mrp/Nbp35 family of cluster scaffolding ATPases form a distinct subfamily within the deviant Walker A family of P-loop NTPases, Dr. Perlstein and her group hypothesize that ATP hydrolysis is utilized to induce a conformational change within the scaffold that is used to coordinate assembly of a nascent cluster on the scaffold with the transfer of this cluster to a recipient. This nucleotide driven switch could impact cluster assembly and/or transfer functions of the scaffold via direct allosteric communication between the cluster scaffolding site and the ATPase site, via regulation of dynamic interactions with other cluster biogenesis proteins, or via a combination of these two mechanisms. By determining how ATPase mutants affect cluster scaffolding activities in vitro, identifying other factors that interact with the scaffold, and elucidating how kinetic mechanism of ATP hydrolysis is affected by scaffolded cluster or other cluster assembly factors, the researchers expect to uncover how nucleotide hydrolysis is exploited by this large family of ATP hydrolyzing iron sulfur cluster scaffolds to coordinate assembly of a nascent cluster within the scaffolding site with the subsequent trafficking steps required to mobilize the cluster from scaffold to target. This research is integrated with an educational plan aimed at exposing K-12 students to research and career opportunities at the chemistry-biology interface via collaboration with Boston University's Learning Resource Network. Furthermore, Professor Perlstein leads the development of an Intensive Biochemistry course for undergraduate chemistry and biology majors tailored to the needs of students planning to pursue research careers.