Iron sulfur (FeS) clusters are ubiquitous metallocofactors required by a large number of proteins involved in myriad cellular processes. Nuclear and cytosolic FeS proteins depend on the cytosolic iron sulfur cluster assembly (CIA) pathway for cluster acquisition. The CIA pathway begins with a scaffolding complex, comprising Nbp35 and Cfd1 in Saccharomyces cerevisiae. Nbp35 and Cfd1 each harbor a deviant Walker A domain for nucleotide hydrolysis that is essential for their FeS cluster scaffolding activity. Since there is little information about the CIA scaffold’s nucleotide hydrolysis activity, it has been challenging to discern the role nucleotide is playing in FeS cluster biogenesis. This thesis investigates the nucleotide driven steps of FeS cluster assembly and transfer, and the individual roles of the scaffold subunits Nbp35 and Cfd1. First addressed was answering the question of why two different scaffold subunits are needed for CIA function, and identifying the scaffold’s quaternary structure. Size exclusion chromatography revealed that the CIA scaffold exists as homodimers and heterodimers. Only Nbp352 and Nbp35-Cfd1 exhibited detectable ATPase activity. Though Cfd12 did not have detectable ATPase activity, it bound nucleotide with an affinity comparable to Nbp352 and Nbp35-Cfd1. Site directed mutagenesis and nucleotide binding studies revealed that the Cfd1 subunit is the high affinity binding site for ATP in Nbp35-Cfd1, and that the Nbp35 subunit binds nucleotide at saturating concentrations. Cfd1 therefore controls nucleotide binding in Nbp35-Cfd1. Additionally, it was found that the Cfd1 subunit is hydrolysis competent when complexed with Nbp35, identifying Nbp35 as an activator of Nbp35-Cfd1’s ATPase activity. Next, ATP’s role in FeS cluster biogenesis by CIA was identified. Mutation of the ATPase domain of Nbp35 impaired the ability of the scaffold to assemble and transfer FeS clusters in vivo. Four phenotypes were identified by observing how each mutation affected the scaffold’s nucleotide binding and hydrolysis. In vitro experiments established that cluster occupancy of the bridging cluster site of Nbp35-Cfd1 decreased the scaffold’s affinity for nucleotide. These results support a model of FeS cluster biogenesis in which nucleotide binding and FeS cluster binding regulate one other, with the bridging cluster site translating information to the ATPase site and vice versa. Nucleotide binding is also proposed to drive a conformational change that mediates interaction with another CIA component, later identified as Dre2. Dre2 was found to stimulate the rate of ATP hydrolysis by Nbp35-Cfd1 in an FeS cluster dependent manner. It is likely that nucleotide hydrolysis is then needed for the scaffold to assemble and/or transfer the FeS cluster. The results of these experiments have allowed us to describe the critical role of nucleotide in FeS biogenesis by CIA and explain the requirement for two distinct scaffold subunits. Finally, a fluorescent [Fe4S4] cluster sensor based on bacterial FNR (fumarate and nitrate reductase transcription factor) was designed, developed, and tested for practicality. FNR was fused to a SNAP tag protein which was then covalently labeled with a fluorescent molecule. The loss of cluster by the sensor resulted in an increase in fluorescence intensity, due to the cluster’s ability to quench fluorescence. As such, cluster decay rates could be measured as a function of increasing fluorescence intensity. The rates observed via fluorescence followed the same trends as the rates obtained by measuring the decay of clusters via absorbance. Encouragingly, the rates observed for the cluster decay were similar to decay rates determined previously via alternative methods.

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