Elucidating the mechanisms of [4Fe-4S] cluster insertions into the cytosolic iron-sulfur protein assembly component Nar1

专门的辅因子运输和插入途径是众多生物合成和代谢反应的重要方面。细胞有不同的机制来确定哪个辅因子插入到哪个蛋白质中，即使在结构相似的蛋白质中也有不同的辅因子占用。大多数金属或生物无机辅助因子转移反应是由蛋白质相互作用控制的，但许多金属蛋白如何将辅助因子从一个蛋白质传递到另一个蛋白质的机制细节尚不清楚，特别是在蛋白质内埋藏的位点。这适用于在细菌，古细菌和真核生物中发现的最普遍的辅助因子类型之一，即铁硫（Fe/S）簇。在模型真核生物酿酒酵母中，18个线粒体铁/S簇（ISC）组装蛋白和11个细胞质铁/S蛋白组装（CIA）组件组成了一个广泛的细胞蛋白质网络，该网络可以合成、运输并将铁/S簇插入目标载脂蛋白中。真核生物中CIA机制的一个有趣方面是，许多运输因子本身结合了多个Fe/S簇，人们对CIA组件本身是如何成熟的仍然知之甚少。例如，Nar1是特殊的，因为它被认为是通过一个不稳定的[4Fe-4S]簇的CIA途径的[4Fe-4S]簇运输中介，同时也是一个需要隐藏的，不可转移的[4Fe-4S]簇的靶载脂蛋白。此外，Nar1与细菌和藻类的[FeFe]-氢化酶同源，但不具有特有的氢化酶功能。为什么氢化酶样蛋白进化成为真核生物铁/S簇运输蛋白仍然是铁/S簇生物发生领域最具挑战性的问题之一。本提案的目的是结合体内、体外和生物物理研究来评估[4Fe-4S]簇插入Nar1的机制，并利用这些信息来确定Nar1在CIA通路中的生理功能。我们将以酿酒酵母为研究对象，研究Nar1的功能以及伴侣结合蛋白在Nar1成熟过程中的重要性。Nar1成熟度的详细信息将指导涉及铁/S簇向Nar1转移和从Nar1转移的重构反应，通过同位素富集的铁/S簇进行量化。通过蛋白质相互作用分析、光谱铁/S聚类分析和x射线晶体结构测定，天然Nar1的生物物理特性也将在一定程度上通过SPP的重要合作进行研究。确定Nar1的分子功能将促进我们对铁/S聚类转移或插入载脂蛋白的多种方式的理解。随后，这也将增强我们对铁/S辅助因子运输中断如何导致铁/S蛋白组装疾病的理解。

Dedicated cofactor trafficking and insertion pathways are an important aspect in a multitude of biosynthetic and metabolic reactions. The cell has various mechanisms for specifically determining which cofactor gets inserted into which protein, even in structurally similar proteins with different cofactor occupancies. A majority of metal or bioinorganic cofactor transfer reactions are governed by protein interactions, yet mechanistic detail remains unclear for many metalloproteins in how they pass cofactors from one protein to the next, especially at sites buried within proteins. This applies to one of the most ubiquitous types of cofactors found in bacteria, archaea, and eukaryotes, namely iron sulfur (Fe/S) clusters. In the model eukaryotic organism Saccharomyces cerevisiae, 18 mitochondrial Fe/S cluster (ISC) assembly proteins and 11 cytosolic Fe/S protein assembly (CIA) components create an extensive cellular protein network, which synthesizes, traffics, and inserts Fe/S clusters into target apoproteins. An interesting aspect of the CIA machinery in eukaryotes is that many of the trafficking factors themselves bind more than one Fe/S cluster and it remains poorly understood how the CIA components themselves are matured. For example, Nar1 is peculiar because it has been proposed to be a [4Fe-4S] cluster trafficking mediator of the CIA pathway via a labile [4Fe-4S] cluster and at the same time a target apoprotein that requires a buried, non-transferable [4Fe-4S] cluster. Furthermore, Nar1 is homologous to bacterial and algal [FeFe]-hydrogenases, but doesn’t have the characteristic hydrogenase function. Why a hydrogenase-like protein has evolved as an essential eukaryotic Fe/S cluster trafficking protein remains one of the most challenging questions to address in the Fe/S cluster biogenesis field. The aim of this proposal is to use a combination of in vivo, in vitro, and biophysical studies to assess mechanisms of [4Fe-4S] cluster insertions into Nar1 and to use this information to determine the physiological function of Nar1 in the CIA pathway. S. cerevisiae will be used as a tractable organism to study the function of Nar1 and the importance of partner binding proteins in Nar1 maturation. Details into Nar1 maturation will guide reconstitution reactions involving Fe/S cluster transfer to and from Nar1, quantified via isotopically-enriched Fe/S clusters. Biophysical characterization of native Nar1 via protein-protein interaction assays, spectroscopic Fe/S cluster analyses, and X-ray crystallographic structure determination will also be pursued, in part, through essential collaborations in the SPP. Assigning the molecular function of Nar1 will advance our understanding of the multiple ways that Fe/S clusters can be transferred or inserted into apoproteins. Subsequently, this will also enhance our understanding of how disruptions in Fe/S cofactor trafficking lead to Fe/S protein assembly diseases.