Iron sulfur (FeS) clusters are ubiquitous cofactors required for numerous fundamental biochemical processes, including DNA replication and repair, transcription, and translation. In the cell, these metallocofactors require a dedicated protein pathway for assembly. The Cytosolic Iron Sulfur Cluster Assembly (CIA) pathway is conserved across higher-level eukaryotes and is responsible for building and inserting these cofactors into the FeS proteins that need them. A major unsolved problem in the FeS cluster biogenesis field is how so many diverse FeS proteins are identified for cluster insertion. Several studies have identified a multiprotein complex containing Cia1, Cia2, and Met18 as the CIA targeting complex responsible for FeS cluster recognition and target maturation. The CIA targeting complex has been shown to associate with an FeS cluster protein, Nar1. Nar1 is a CIA factor that plays an unknown role in cluster transfer. Little information is known about the structure of the CIA targeting complex its mechanism of FeS cluster protein recognition. In this thesis, I investigate the architecture of the CIA targeting complex as well as the role each subunit plays in identification of apo-proteins and iron-sulfur cluster insertion. Previous proteomic and cell biological studies from the Lill lab propose that the CIA targeting complex exists as a mixture of discrete complexes in vivo. Each of these complexes is responsible for recognizing a distinct subset of targets. Herein, we utilize affinity co-purification and size exclusion chromatography investigate connectivity of the targeting complex, identify stable subcomplexes, and define their roles in recognizing our two model targets Rad3 and Leu1. We determine the CIA targeting complex contains one Met18, two Cia1, and four Cia2 polypepides. This complex is required to recognize Leu1. Our experiments reveal the formation of the stable subcomplexes Cia1-Cia2 and Met18-Cia2, which is sufficient to identify to Rad3. We also interrogate the role of Nar1 in binding to targets and cluster transfer, excluding the model that it acts as an adapter for cluster transfer. Furthermore, using site directed mutagenesis, combined with our co-purification and in vivo assays, we map the key interfaces required to form the targeting complex and investigate how their mutations impacts CIA function in vivo. We identify the binding site of Cia1 on Cia2, as well as the general region in which Cia2 binds to Met18. Through these experiments, we shed light on the role these subunits of CIA targeting complex and Nar1 play in FeS target recognition and FeS cluster transfer.

Complex biosynthetic pathways are required for the assembly and insertion of iron-sulfur (Fe-S) cluster cofactors. The four cluster biogenesis systems that have been discovered require at least one ATPase, but generally the function of nucleotide hydrolysis is understudied. In the cytosolic iron sulfur cluster assembly (CIA) system, responsible for delivering [Fe4-S4] cluster cofactors for cytosolic and nuclear enzymes, the assembly scaffold comprises two homologous ATPases, called Nbp35 and Cfd1 in Saccharomyces cerevisiae. Genetic studies have discovered that the ATPase sites are required for scaffold function in vivo, but in vitro studies have failed to reveal why. The ATPase sites of the Nbp35 and Cfd1 contain a conserved P-loop nucleotide-binding protein fold with a deviant Walker A motif. Known metal trafficking P-loop NTPases’ metallochaperone mechanisms rely on both nucleotide binding and hydrolysis to properly assemble and deliver metal cargo. Furthermore, P-loop NTPases with a deviant Walker A motif commonly serve as central regulatory switches whose hydrolysis activity is modulated by small molecule cargos and/or protein partners. Therefore, it is proposed that the role of Nbp35-Cfd1’s ATPase sites is to direct Fe-S cluster movement by regulating protein and metal cargo interactions. The goal of this thesis is to better understand the scaffold reaction cycle by investigating the metallochaperone mechanism through Nbp35-Cfd1’s protein communications with its ATPase sites. To do this, the identification of at least one nucleotide-dependent partner protein must first be discovered. Herein, in vitro methods have been developed to uncover the scaffold’s ATPase site regulation of protein interactions. We describe a qualitative affinity copurification assay and a quantitative analysis for evaluating the dissociation constant and the kcat and Km values for ATP hydrolysis for the scaffold–partner protein complex. Additionally, the execution of these ATPase assays in an anaerobic environment can be applied to study nucleotide hydrolases involved in metallocluster biogenesis. These in vitro methods are applied to Nbp35-Cfd1 and it is discovered that ATP binding and hydrolysis regulates Nbp35-Cfd1 binding with two CIA factors: Dre2, a reductase proposed to assist in Fe-S cluster assembly, and Nar1, an adaptor between the early and late CIA factors. Although reconstitution of the scaffold’s Fe-S clusters results in a two-fold increase in its ATPase activity, the Dre2 and Nar1 ATP hydrolysis stimulation is dampened, demonstrating that both the Fe-S cargo and partner proteins regulate the scaffold’s ATPase reaction cycle. Next, the domains required for binding and ATPase stimulation were identified for Nbp35-Cfd1 with its partner proteins Dre2 and Nar1. The C-terminal Fe-S binding domain of Dre2 is sufficient for ATPase stimulation, while the Nar1 requires both its N- and C-terminal Fe-S binding domains to activate Nbp35-Cfd1’s ATP hydrolysis. The N-terminal Fe-S binding domain of Nbp35 is dispensable for binding and ATPase stimulation of both Dre2 and Nar1. The CIA targeting complex protein Cia1, which binds to Nar1, competes off Nbp35-Cfd1, indicating a shared binding domain. This data both validates and refines the current working model of the CIA system. To test whether the communication between the ATPase and Fe-S cluster binding domains of the CIA scaffold functions in an analogous manner across multiple species, a preliminary analysis was completed for whether Chaetomium thermophilum and Homo sapien Nbp35-Cfd1 exhibit similar ATPase characteristics and partner protein interaction as their S. cerevisiae ortholog. Human and fungal Nbp35-Cfd1 exhibit ATP binding and demonstrate nucleotide-dependent interactions with Dre2 and Nar1, suggesting that these interactions in a similar manner to effectively communicate in the CIA pathway. Overall, our study uncovers striking similarities between the CIA pathway and other systems which exploit a deviant Walker A NTPase to coordinate complex, multiprotein processes. Identification of the scaffold’s partner proteins significantly advances our understanding as to why the Nbp35/MRP-type Fe-S cluster biogenesis proteins are nucleotide hydrolases. This work provides some mechanistic insight into the functions of these proteins and provides a roadmap for how to investigate this large and widely distributed family and other P-loop NTPase metallochaperones. / 2024-09-19T00:00:00Z