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

Abstract

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 Better Survival of MSI Subtype Is Associated With the Oxidative Stress Related Pathways in Gastric Cancer

“…NUBP2, the nucleotide binding protein 2, encodes adenosine triphosphate (ATP) and metal-binding protein that modulate the iron-metabolism that was essential for ATP production and mitochondrial metabolism (48). It was the essential component that could assemble the iron-sulfur clusters through the process of cytosolic iron-sulfur cluster assembly (CIA) outside of the mitochondria (49,50). Compared with the hypoxic microenvironment, the upregulation of NUBP2 indicated the normoxia and ensure the oxidative phosphorylation in the MSI samples in GC.…”

A review of TNP-ATP in protein binding studies: benefits and pitfalls

“…Grossman et al. ( 37 ) used TNP fluorescence to determine the K d of the Nbp35-Cfd1 complex for TNP-ATP and then used fluorescence anisotropy to determine the K d for two other fluorescently labeled ATPs: MANT-ATP (N-methylanthraniloyl-ATP) and BoATP (BODIPY-FL 2′/3′-O-[N-(2-aminoethyl)urethane] ATP). They report a K d for TNP-ATP that is more than 100× tighter than the K d for either MANT-ATP or BoATP ( Table 1 ).…”

“…Although all will have the same kind of problems, e.g., an extra group that will enhance or interfere with ATP binding, each will do so differently and provide a broader view of the interaction of ATP with proteins. Many fluorescent tags have been made and attached to either ATP (e.g., ε ATP or etheno-ATP) or other nucleotides; one more commonly used tag is MANT (N-methylanthraniloyl) ( 8 , 24 , 37 , 78 , 129 , 130 , 131 , 132 , 133 ). Whereas ε ATP is modified on the adenosine ring of ATP, MANT and TNP are attached to the ribose sugar.…”

Unraveling the mechanism of [4Fe‐4S] cluster assembly on the N‐terminal cluster binding site of NUBP1

“…One of these is NUBP1 (Camponeschi et al, 2020 ), another component of the CIA machinery, acting as a scaffold for the assembly of [4Fe‐4S] clusters together with its homologous NUBP2 (Stehling et al, 2008 ; Stehling et al, 2018 ). NUBP1 and NUBP2 belong to the deviant Walker A family NTPases (Grossman et al, 2019 ), and are both required for the CIA machinery (Stehling et al, 2008 ; Stehling et al, 2018 ). The two proteins form homo‐ and heterodimeric complexes, bridging a [4Fe‐4S] cluster bound to a CxxC motif close to the C‐termini of both proteins (Netz et al, 2012 ; Stehling et al, 2018 ).…”