Coupling Nucleotide Binding and Hydrolysis to Iron-Sulfur Cluster Acquisition and Transfer Revealed through Genetic Dissection of the Nbp35 ATPase Site.

The cytosolic iron-sulfur cluster assembly (CIA) scaffold, comprising Nbp35 and Cfd1 in yeast, assembles iron-sulfur (FeS) clusters destined for cytosolic and nuclear enzymes. ATP hydrolysis by the CIA scaffold plays an essential but poorly understood role in cluster biogenesis. Here we find that mutation of conserved residues in the four motifs comprising the ATPase site of Nbp35 diminished the scaffold's ability to both assemble and transfer its FeS cluster in vivo. The mutants fall into four phenotypic classes that can be understood by how each set of mutations affects ATP binding and hydrolysis. In vitro studies additionally revealed that occupancy of the bridging FeS cluster binding site decreases the scaffold's affinity for the nucleotide. On the basis of our findings, we propose that nucleotide binding and hydrolysis by the CIA scaffold drive a series of protein conformational changes that regulate association with other proteins in the pathway and with its newly formed FeS cluster. Our results provide insight into how the ATPase and cluster scaffolding activities are allosterically integrated.

The Cfd1 Subunit of the Nbp35-Cfd1 Iron Sulfur Cluster Scaffolding Complex Controls Nucleotide Binding.

The cytosolic iron sulfur cluster assembly (CIA) scaffold biosynthesizes iron sulfur cluster cofactors for enzymes residing in the cytosol and the nucleus. In fungi and animals, it comprises two homologous ATPases, called Nbp35 and Cfd1 in yeast, which can form homodimeric and heterodimeric complexes. Both proteins are required for CIA function, but their individual roles are not well understood. Here we investigate the nucleotide affinity of each form of the scaffold for ATP and ADP to reveal any differences that could shed light on the functions of the different oligomeric forms of the protein or any distinct roles of the individual subunits. All forms of the CIA scaffold are specific for adenosine nucleotides and not guanosine nucleotides. Although the Cfd1 homodimer has no detectable ATPase activity, it binds ATP with an affinity comparable to that of the hydrolysis competent forms, Nbp352 and Nbp35-Cfd1. Titrations to determine the number of nucleotide binding sites combined with site-directed mutagenesis demonstrate that the nucleotide must bind to the Cfd1 subunit of the heterodimer before it can bind to Nbp35 and that the Cfd1 subunit is hydrolysis competent when bound to Nbp35 in the heterodimer. Altogether, our work reveals the distinct roles of the Nbp35 and Cfd1 subunits in their heterodimeric complex. Cfd1 controls nucleotide binding, and the Nbp35 subunit is required to activate nucleotide hydrolysis.

Approaches to Interrogate the Role of Nucleotide Hydrolysis by Metal Trafficking NTPases: The Nbp35-Cfd1 Iron-Sulfur Cluster Scaffold as a Case Study.

Nucleotide hydrolases play integral yet poorly understood roles in several metallocluster biosynthetic pathways. For example, the cytosolic iron-sulfur cluster assembly (CIA) is initiated by the CIA scaffold, an ATPase which builds new iron-sulfur clusters for proteins localized to the cytosol and the nucleus in eukaryotic organisms. While in vivo studies have demonstrated the scaffold's nucleotide hydrolase domain is vital for its function, in vitro approaches have not revealed tight allosteric coupling between the cluster scaffolding site and the ATPase site. Thus, the role of ATP hydrolysis has been hard to pinpoint. Herein, we describe methods to probe the nucleotide affinity and hydrolysis activity of the CIA scaffold from yeast, which is comprised of two homologous polypeptides called Nbp35 and Cfd1. In particular, we report two different equilibrium binding assays that make use of commercially available fluorescent nucleotide analogs. Importantly, these assays can be applied to probe nucleotide affinity of both the apo- and holo-forms of the CIA scaffold. Generally, these fluorescent nucleotide analogs have been underutilized to probe metal trafficking NTPase because one of the most commonly used probes, mantATP, which is labeled with the methylanthraniloyl probe via the 2' or 3' sugar hydroxyls, has an absorption which overlaps with the UV-Vis features of many metal-binding proteins. However, by exploiting analogs like BODIPY-FL and trinitrophenyl-labeled nucleotides which have better photophysical properties for metalloprotein applications, these approaches have the potential to reveal the mechanistic underpinnings of NTPases required for metallocluster biosynthesis.

Defining the domains of Cia2 required for its essential function in vivo and in vitro.

The cytosolic iron-sulfur cluster assembly (CIA) system biosynthesizes iron-sulfur (FeS) cluster cofactors for cytosolic and nuclear proteins. The yeast Cia2 protein is the central component of the targeting complex which identifies apo-protein targets in the final step of the pathway. Herein, we determine that Cia2 contains five conserved motifs distributed between an intrinsically disordered N-terminal domain and a C-terminal domain of unknown function 59 (DUF59). The disordered domain is dispensible for binding the other subunits of the targeting complex, Met18 and Cia1, and the apo-target Rad3 in vitro. While in vivo assays reveal that the C-terminal domain is sufficient to support viability, several phenotypic assays indicate that deletion of the N-terminal domain negatively impacts CIA function. We additionally establish that Glu208, located within a conserved motif found only in eukaryotic DUF59 proteins, is important for the Cia1-Cia2 interaction in vitro. In vivo, E208A-Cia2 results in a diminished activity of the cytosolic iron sulfur cluster protein, Leu1 but only modest effects on hydroxyurea or methylmethane sulfonate sensitivity. Finally, we demonstrate that neither of the two highly conserved motifs of the DUF59 domain are vital for any of Cia2's interactions in vitro yet mutation of the DPE motif in the DUF59 domain results in a nonfunctional allele in vivo. Our observation that four of the five highly conserved motifs of Cia2 are dispensable for targeting complex formation and apo-target binding suggests that Cia2 is not simply a protein-protein interaction mediator but it likely possesses an additional, currently cryptic, function during the final cluster insertion step of CIA.

The Yeast Nbp35-Cfd1 Cytosolic Iron-Sulfur Cluster Scaffold Is an ATPase.

Nbp35 and Cfd1 are prototypical members of the MRP/Nbp35 class of iron-sulfur (FeS) cluster scaffolds that function to assemble nascent FeS clusters for transfer to FeS-requiring enzymes. Both proteins contain a conserved NTPase domain that genetic studies have demonstrated is essential for their cluster assembly activity inside the cell. It was recently reported that these proteins possess no or very low nucleotide hydrolysis activity in vitro, and thus the role of the NTPase domain in cluster biogenesis has remained uncertain. We have reexamined the NTPase activity of Nbp35, Cfd1, and their complex. Using in vitro assays and site-directed mutagenesis, we demonstrate that the Nbp35 homodimer and the Nbp35-Cfd1 heterodimer are ATPases, whereas the Cfd1 homodimer exhibited no or very low ATPase activity. We ruled out the possibility that the observed ATP hydrolysis activity might result from a contaminating ATPase by showing that mutation of key active site residues reduced activity to background levels. Finally, we demonstrate that the fluorescent ATP analog 2'/3'-O-(N'-methylanthraniloyl)-ATP (mantATP) binds stoichiometrically to Nbp35 with a KD = 15.6 µM and that an Nbp35 mutant deficient in ATP hydrolysis activity also displays an increased KD for mantATP. Together, our results demonstrate that the cytosolic iron-sulfur cluster assembly scaffold is an ATPase and pave the way for interrogating the role of nucleotide hydrolysis in cluster biogenesis by this large family of cluster scaffolding proteins found across all domains of life.