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Iron–sulfur (Fe/S) clusters are essential protein cofactors crucial for many cellular functions including DNA maintenance, protein translation, and energy conversion. De novo Fe/S cluster synthesis occurs on the mitochondrial scaffold protein ISCU and requires cysteine desulfurase NFS1, ferredoxin, frataxin, and the small factors ISD11 and ACP (acyl carrier protein). Both the mechanism of Fe/S cluster synthesis and function of ISD11-ACP are poorly understood. Here, we present crystal structures of three different NFS1-ISD11-ACP complexes with and without ISCU, and we use SAXS analyses to define the 3D architecture of the complete mitochondrial Fe/S cluster biosynthetic complex. Our structural and biochemical studies provide mechanistic insights into Fe/S cluster synthesis at the catalytic center defined by the active-site Cys of NFS1 and conserved Cys, Asp, and His residues of ISCU. We assign specific regulatory rather than catalytic roles to ISD11-ACP that link Fe/S cluster synthesis with mitochondrial lipid synthesis and cellular energy status. Fe/S clusters are synthesized by the mitochondrial iron-sulfur cluster assembly (ISC) machinery. Here the authors combine crystallography and small angle X-ray scattering measurements to structurally characterize the core ISC complex and give functional insights into eukaryotic Fe/S cluster synthesis.

The core machinery for de novo biosynthesis of iron-sulfur clusters (ISC), located in the mitochondria matrix, is a five-protein complex containing the cysteine desulfurase NFS1 that is activated by frataxin (FXN), scaffold protein ISCU, accessory protein ISD11, and acyl-carrier protein ACP. Deficiency in FXN leads to the loss-of-function neurodegenerative disorder Friedreich’s ataxia (FRDA). Here the 3.2 Å resolution cryo-electron microscopy structure of the FXN-bound active human complex, containing two copies of the NFS1-ISD11-ACP-ISCU-FXN hetero-pentamer, delineates the interactions of FXN with other component proteins of the complex. FXN binds at the interface of two NFS1 and one ISCU subunits, modifying the local environment of a bound zinc ion that would otherwise inhibit NFS1 activity in complexes without FXN. Our structure reveals how FXN facilitates ISC production through stabilizing key loop conformations of NFS1 and ISCU at the protein–protein interfaces, and suggests how FRDA clinical mutations affect complex formation and FXN activation. The iron-sulfur cluster (ISC) assembly complex is activated by frataxin (FXN) and Friedreich’s ataxia is caused by FXN deficiency. Here the authors present the 3.2 Å resolution cryo-EM structure of the human frataxin bound ISC complex and discuss how FXN activates enzymatic activity.

Iron-sulfur (FeS) protein biogenesis in eukaryotes begins with the de novo assembly of [2Fe-2S] clusters by the mitochondrial core iron-sulfur cluster assembly (ISC) complex. This complex comprises the scaffold protein ISCU2, the cysteine desulfurase subcomplex NFS1-ISD11-ACP1, the allosteric activator frataxin (FXN) and the electron donor ferredoxin-2 (FDX2). The structural interaction of FDX2 with the complex remains unclear. Here, we present cryo-EM structures of the human FDX2-bound core ISC complex showing that FDX2 and FXN compete for overlapping binding sites. FDX2 binds in either a ‘distal’ conformation, where its helix F interacts electrostatically with an arginine patch of NFS1, or a ‘proximal’ conformation, where this interaction tightens and the FDX2-specific C terminus binds to NFS1, facilitating the movement of the [2Fe-2S] cluster of FDX2 closer to the ISCU2 FeS cluster assembly site for rapid electron transfer. Structure-based mutational studies verify the contact areas of FDX2 within the core ISC complex. De novo [2Fe-2S] cluster synthesis by the mitochondrial ISC complex requires electrons from ferredoxin-2 (FDX2). The authors map FDX2 binding to the core ISC complex, showing that FDX2 can bind in two possible conformations in a manner competitive with frataxin.

Friedreich’s ataxia is a rare, progressive, genetic disorder, the root cause of which is a significant deficiency in the mitochondrial protein frataxin. Frataxin is ubiquitously expressed, but its deficiency results in a variety of debilitating symptoms, with disease severity, rate of progression and age of onset inversely correlating with tissue frataxin levels. Nomlabofusp is a novel cell penetrant peptide based recombinant fusion protein designed to enter cells and deliver human FXN into the mitochondria. Using immunofluorescence staining and western blot we show that frataxin delivered by nomlabofusp is detected in the mitochondria of H9c2 and SH-SY5Y cells. Also in these cells, and in C2C12 and HEK293 cells, we demonstrate the presence of mature frataxin after nomlabofusp exposure. Finally, using buccal swab tissue samples taken from study subjects in a Phase 1 clinical trial who received nomlabofusp, we show increases in mature frataxin levels along with marked changes in gene expression post-administration suggesting intracellular pharmacodynamic activity. Together, these results demonstrate that nomlabofusp enters the cell and localizes to the mitochondria, releasing mature frataxin that appears to be biologically active and support the use of nomlabofusp as a potential treatment for patients with Friedreich’s ataxia. Graphical Abstract

Folding and function may impose different requirements on the amino acid sequences of proteins, thus potentially giving rise to conflict. Such a conflict, or frustration, can result in the formation of partially misfolded intermediates that can compromise folding and promote aggregation. We investigate this phenomenon by studying frataxin, a protein whose normal function is to facilitate the formation of iron-sulfur clusters but whose mutations are associated with Friedreich's ataxia. To characterize the folding pathway of this protein we carry out a Φ-value analysis and use the resulting structural information to determine the structure of the folding transition state, which we then validate by a second round of rationally designed mutagenesis. The analysis of the transition-state structure reveals that the regions involved in the folding process are highly aggregation-prone. By contrast, the regions that are functionally important are partially misfolded in the transition state but highly resistant to aggregation. Taken together, these results indicate that in frataxin the competition between folding and function creates the possibility of misfolding, and that to prevent aggregation the amino acid sequence of this protein is optimized to be highly resistant to aggregation in the regions involved in misfolding.

Friedreich’s ataxia is an incurable genetic disorder caused by a mutant expansion of the trinucleotide GAA within an intronic FXN RNA. This expansion leads to reduced expression of frataxin (FXN) protein and evidence suggests that transcriptional repression is caused by an R-loop that forms between the expanded repeat RNA and complementary genomic DNA. Synthetic agents that increase levels of FXN protein might alleviate the disease. We demonstrate that introducing anti-GAA duplex RNAs or single-stranded locked nucleic acids into patient-derived cells increases FXN protein expression to levels similar to analogous wild-type cells. Our data are significant because synthetic nucleic acids that target GAA repeats can be lead compounds for restoring curative FXN levels. More broadly, our results demonstrate that interfering with R-loop formation can trigger gene activation and reveal a new strategy for upregulating gene expression. Expansion of the trinucleotide GAA within an intronic FXN RNA can cause Friedreich's Ataxia (FRDA), an incurable genetic disorder. Here, the authors show that anti-GAA duplex RNAs or single-stranded locked nucleic acids increases FXN protein expression in patient-derived cells to levels similar to wild-type cells.

Reduced levels of frataxin, an essential protein of as yet unknown function, are responsible for causing the neurodegenerative pathology Friedreich's ataxia. Independent reports have linked frataxin to iron–sulphur cluster assembly through interactions with the two central components of this machinery: desulphurase Nfs1/IscS and the scaffold protein Isu/IscU. In this study, we use a combination of biophysical methods to define the structural bases of the interaction of CyaY (the bacterial orthologue of frataxin) with the IscS/IscU complex. We show that CyaY binds IscS as a monomer in a pocket between the active site and the IscS dimer interface. Recognition does not require iron and occurs through electrostatic interactions of complementary charged residues. Mutations at the complex interface affect the rates of enzymatic cluster formation. CyaY binding strengthens the affinity of the IscS/IscU complex. Our data suggest a new paradigm for understanding the role of frataxin as a regulator of IscS functions. Frataxin is an essential protein that has been linked to iron–sulphur cluster assembly, and reduced levels are associated with Friedrich's ataxia. In this study, a combination of techniques is used to probe the interactions of the bacterial frataxin orthologue CyaY with the iron–sulphur cluster assembly machinery.

Many bacteria contain cytoplasmic chemoreceptors that lack sensor domains. Here, we demonstrate that such cytoplasmic receptors found in 8 different bacterial and archaeal phyla genetically couple to metalloproteins related to β-lactamases and nitric oxide reductases. We show that this oxygen-binding di-iron protein (ODP) acts as a sensor for chemotactic responses to both iron and oxygen in the human pathogen Treponema denticola (Td). The ODP di-iron site binds oxygen at high affinity to reversibly form an unusually stable μ-peroxo adduct. Crystal structures of ODP from Td and the thermophile Thermotoga maritima (Tm) in the Fe[III]₂-O₂2−, Zn[II], and apo states display differences in subunit association, conformation, and metal coordination that indicate potential mechanisms for sensing. In reconstituted systems, iron-peroxo ODP destabilizes the phosphorylated form of the receptor-coupled histidine kinase CheA, thereby providing a biochemical link between oxygen sensing and chemotaxis in diverse prokaryotes, including anaerobes of ancient origin.

Friedreich ataxia is an autosomal recessive neurodegenerative disease associated with a high diabetes prevalence. No treatment is available to prevent or delay disease progression. Friedreich ataxia is caused by intronic GAA trinucleotide repeat expansions in the frataxin-encoding FXN gene that reduce frataxin expression, impair iron-sulfur cluster biogenesis, cause oxidative stress, and result in mitochondrial dysfunction and apoptosis. Here we examined the metabolic, neuroprotective, and frataxin-inducing effects of glucagon-like peptide-1 (GLP-1) analogs in in vivo and in vitro models and in patients with Friedreich ataxia. The GLP-1 analog exenatide improved glucose homeostasis of frataxin-deficient mice through enhanced insulin content and secretion in pancreatic β cells. Exenatide induced frataxin and iron-sulfur cluster–containing proteins in β cells and brain and was protective to sensory neurons in dorsal root ganglia. GLP-1 analogs also induced frataxin expression, reduced oxidative stress, and improved mitochondrial function in Friedreich ataxia patients’ induced pluripotent stem cell–derived β cells and sensory neurons. The frataxin-inducing effect of exenatide was confirmed in a pilot trial in Friedreich ataxia patients, showing modest frataxin induction in platelets over a 5-week treatment course. Taken together, GLP-1 analogs improve mitochondrial function in frataxin-deficient cells and induce frataxin expression. Our findings identify incretin receptors as a therapeutic target in Friedreich ataxia.

Frataxin is a highly conserved protein encoded by the frataxin ( FXN ) gene. The full-length 210-amino acid form of protein frataxin (1–210; isoform A) expressed in the cytosol of cells rapidly translocates to the mitochondria, where it is converted to the mature form (81–210) by mitochondrial processing peptidase. Mature frataxin (81–210) is a critically important protein because it facilitates the assembly of mitochondrial iron-sulfur cluster protein complexes such as aconitase, lipoate synthase, and succinate dehydrogenases. Decreased expression of frataxin protein is responsible for the devastating rare genetic disease of Friedreich’s ataxia. The mitochondrial form of frataxin has long been thought to be present in erythrocytes even though paradoxically, erythrocytes lack mitochondria. We have discovered that erythrocyte frataxin is in fact a novel isoform of frataxin (isoform E) with 135-amino acids and an N-terminally acetylated methionine residue. There is three times as much isoform E in erythrocytes (20.9 ± 6.4 ng/mL) from the whole blood of healthy volunteers (n = 10) when compared with the mature mitochondrial frataxin present in other blood cells (7.1 ± 1.0 ng/mL). Isoform E lacks a mitochondrial targeting sequence and so is distributed to both cytosol and the nucleus when expressed in cultured cells. When extra-mitochondrial frataxin isoform E is expressed in HEK 293 cells, it is converted to a shorter isoform identical to the mature frataxin found in mitochondria, which raises the possibility that it is involved in disease etiology. The ability to specifically quantify extra-mitochondrial and mitochondrial isoforms of frataxin in whole blood will make it possible to readily follow the natural history of diseases such as Friedreich’s ataxia and monitor the efficacy of therapeutic interventions.