Leveraging FIDA to monitor competitive binding during Fe-S cluster synthesis.

Abstract

Iron-Sulfur (Fe-S) clusters constitute ancient cofactors that accompany a versatile range of fundamental biological reactions across eukaryotes and prokaryotes. Mitochondrial eukaryotic [2Fe-2S] cluster synthesis is coordinated by the Iron-Sulfur Cluster (ISC) machinery which encompasses a core (NIAU2) scaffold that interacts with additional proteins FXN and FDX2 during Fe-S biogenesis. Here, FIDA was used to monitor the binding of FXN and FDX2 to the (NIAU2) complex and was further leveraged to validate and characterize a mutually exclusive mode of binding. Following this, a predictive model of the (NIAU-FDX2) complex was used to design a series of FDX2 mutant constructs to better understand the mechanism binding and its corresponding affinity constants were determined by FIDA competition assays. Through this, we mapped residues critical for the interaction of FDX2 with the (NIAU2) complex and further discovered that high levels of FDX2 disrupts [2Fe-2S] cluster synthesis through both competition with FXN and an auto-inhibitory mechanism involving its C-terminal tail. Taken together, our data suggests that targeting FDX2 in the context of FXN deficiency, as is present in the related disease Friedreich’s ataxia (FRDA), may constitute a novel therapeutic avenue.

Efficient [2Fe-2S] synthesis requires a balanced ratio of Ferredoxin-2 and Frataxin

Abstract

Iron-sulfur (FeS) clusters are indispensable molecular entities involved in crucial cellular processes across all domains of life. They participate in energy metabolism, DNA repair, and gene expression regulation in eukaryotes, with common clusters including [2Fe2S], [4Fe4S], and [4Fe3S]. Mammalian FeS cluster generation primarily relies on the Iron-Sulfur Cluster (ISC) machinery in mitochondria, which consists of a core NFS1-ISD11-ACP-ISCU interacting with the external proteins FXN and FDX2 during [2Fe2S] synthesis. Dysregulation in this process, as seen in Friedreich’s ataxia (FA), due to decreased FXN expression, results in FeS cluster shortage and subsequent cardiac impairment. Therapeutic strategies being developed for FA involve AAV-mediated gene therapy to regenerate FXN levels, yet challenges persist due to toxicity associated with FXN overexpression. Our study investigates the competitive binding interactions of FXN and FDX2 in the mammalian ISC complex, which we confirm through binding between FXN and FDX2 in the mammalian ISC complex, which we confirm through in vitro FeS cluster assembly. Using biochemical assays, we demonstrate that modulating FDX2 can mitigate FXN-associated toxicity while also enhancing FeS cluster synthesis efficiency, offering insights for FA therapeutic development. This work highlights the intricate balance required for optimal FeS cluster synthesis and proposes novel strategies for managing the FXN-associated FA disorder.

This video includes my flash talk for the 2023 Friedreich's ataxia research alliance (FARA) Flash Talk Series. This talk begins at 16:37, and was subsequently awarded as the highest rated talk (awarded $100).