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

专用的辅因子运输和插入途径是许多生物合成和代谢反应的重要方面。细胞有不同的机制来具体确定哪种辅因子被插入到哪种蛋白质中，即使在结构相似的蛋白质中也是如此，但辅因子所占的比例不同。大多数金属或生物无机辅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因子从一个蛋白质传递到下一个蛋白质的机制细节仍不清楚，特别是在蛋白质中埋藏的位置。这适用于在细菌、古菌和真核生物中发现的最普遍的辅因子类型之一，即铁硫(Fe/S)簇。在模式真核生物酿酒酵母中，18个线粒体Fe/S簇(ISC)组装蛋白和11个胞质Fe/S蛋白组装(CIA)组分形成了一个广泛的细胞蛋白质网络，合成、运输并将Fe/S簇插入到目标脱辅基蛋白中。在真核生物中，CIA机制的一个有趣的方面是，许多贩运因子本身结合了不止一个Fe/S簇，而对于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CIA途径的[4Fe-4S]簇运输媒介，通过不稳定的[4Fe-4S]簇，同时是需要掩埋的、不可转移的[4Fe-4S]簇的靶标载脂蛋白。此外，Nar1与细菌和藻类的[FeFe]-氢酶同源，但不具有特有的氢酶功能。为什么氢酶样蛋白进化为真核生物铁/S簇运输蛋白，仍然是铁/S簇生物发生领域中最具挑战性的问题之一。该建议的目的是结合体内、体外和生物物理研究来评估[4Fe-4S]簇插入到Nar1的机制，并利用这些信息来确定Nar1在CIA途径中的生理功能。酿酒酵母将作为一种易驯化的生物来研究Nar1的功能以及伴侣结合蛋白在Nar1成熟过程中的重要性。关于NAR1成熟度的细节将指导涉及Fe/S团簇来往于NAR1的重构反应，通过同位素富集态的Fe/S团簇进行量化。还将通过蛋白质-蛋白质相互作用分析、光谱分析Fe/S簇分析和X射线晶体结构测定，部分通过SPP中的基本合作来进行天然Nar1的生物物理表征。确定NAR1的分子功能将有助于我们理解Fe/S簇转移或插入到脱脂蛋白中的多种途径。随后，这也将加深我们对铁/S辅因子转运中断如何导致铁/S蛋白组装病的理解。

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.