Iron-sulfur Cluster Biosynthesis Research Articles

PP03.14 – 2978: Massive early leukoencephalopathy caused by a pathogenic mutation in IBA57: A new clinical presentation for this recently described gene defect

Objective Unraveling the underlying molecular defect in a patient with early onset progressive leukoencephalopathy and combined OXPHOS deficiency involving complex I and II. Methods After homozygosity mapping and whole exome sequencing a candidate gene was identified. Pathogenicity was assessed by means of functional studies in HeLa cells and western blotting in patient material. Results In a patient presenting with psychomotor retardation, massive white matter alterations and the presence of high lactate were detected on MRI, and a combined complex I and II deficiency was found in skeletal muscle. This combined complex deficiency is very suggestive of a defective iron-sulfur cluster (ISC) biosynthesis, a suspicion which was further strengthened by absence of lipoic residues in aKGDH and PDH. Homozygosity mapping revealed several candidate genes involved in ISC biogenesis: NFU1, BOLA3, IBA57 and ABCB10. Whole exome sequencing identified a homozygous variant in IBA57 [c.436C> T. (p.Arg146Trp)]. Complementation studies in HeLa cells depleted of wild type IBA57 and transfected with mutant IBA57 showed deficient lipoylation and lowered expression of a complex II subunit. Conclusion By combining a biochemical and molecular approach the underlying genetic cause for this patient's phenotype could be unraveled. This is the third report of a pathogenic variant in IBA57 and discloses the clinical and neuroradiological heterogeneity of this gene defect. At the biochemical level a combined complex I and II deficiency and absence of lipoylation seems to be a uniform finding.

Selenate reductase activity inEscherichia colirequires Isc iron-sulfur cluster biosynthesis genes

The selenate reductase in Escherichia coli is a multi-subunit enzyme predicted to bind Fe-S clusters. In this study, we examined the iron-sulfur cluster biosynthesis genes that are required for selenate reductase activity. Mutants devoid of either the iscU or hscB gene in the Isc iron-sulfur cluster biosynthesis pathway lost the ability to reduce selenate. Genetic complementation by the wild-type sequences restored selenate reductase activity. The results indicate the Isc biosynthetic system plays a key role in selenate reductase Fe-S cofactor assembly and is essential for enzyme activity.

PH-induced conformational change of IscU at low pH correlates with protonation/deprotonation of two conserved histidine residues.

IscU, the scaffold protein for the major iron–sulfur cluster biosynthesis pathway in microorganisms and mitochondria (ISC pathway), plays important roles in the formation of [2Fe–2S] and [4Fe–4S] clusters and their delivery to acceptor apo-proteins. Our laboratory has shown that IscU populates two distinct, functionally relevant conformational states, a more structured state (S) and a more dynamic state (D), that differ by cis/trans isomerizations about two peptidyl-prolyl peptide bonds [Kim, J. H., Tonelli, M., and Markley, J. L. (2012) Proc. Natl. Acad. Sci. U.S.A., 109, 454–459. Dai Z., Tonelli, M., and Markley, J. L. (2012) Biochemistry, 51, 9595–9602. Cai, K., Frederick, R. O., Kim, J. H., Reinen, N. M., Tonelli, M., and Markley, J. L. (2013) J. Biol. Chem., 288, 28755–28770]. Here, we report our findings on the pH dependence of the D ⇄ S equilibrium for Escherichia coli IscU in which the D-state is stabilized at low and high pH values. We show that the lower limb of the pH dependence curve results from differences in the pKa values of two conserved histidine residues (His10 and His105) in the two states. The net proton affinity of His10 is about 50 times higher and that of His105 is 13 times higher in the D-state than in the S-state. The origin of the high limb of the D ⇄ S pH dependence remains to be determined. These results show that changes in proton inventory need to be taken into account in the steps in iron–sulfur cluster assembly and transfer that involve transitions of IscU between its S- and D-states.

Cysteine desulphurase plays an important role in environmental adaptation of the hyperthermophilic archaeon Thermococcus kodakarensis.

The sulphur atoms of sulphur-containing cofactors that are essential for numerous cellular functions in living organisms originate from L-cysteine via cysteine desulphurase (CSD) activity. However, many (hyper)thermophilic archaea, which thrive in solfataric fields and are positioned near the root of the evolutionary tree of life, lack CSD orthologues. The existence of CSD orthologues in a subset of (hyper)thermophilic archaea is of interest with respect to the evolution of sulphur-trafficking systems for the cofactors. This study demonstrates that the disruption of the csd gene of Thermococcus kodakarensis, a facultative elemental sulphur (S(0))-reducing hyperthermophilic archaeon, encoding Tk-CSD, conferred a growth defect evident only in the absence of S(0), and that growth can be restored by the addition of S(0), but not sulphide. We show that the csd gene is not required for biosynthesis of thiamine pyrophosphate or molybdopterin, irrespective of the presence or absence of S(0), but is necessary for iron-sulphur cluster biosynthesis in the absence of S(0). Recombinant form of Tk-CSD expressed in Escherichia coli was obtained and it was found to catalyse the desulphuration of L-cysteine. The obtained data suggest that hyperthermophiles might benefit from a capacity for CSD-dependent iron-sulphur cluster biogenesis, which allows them to thrive outside solfataric environments.

The unique regulation of iron-sulfur cluster biogenesis in a Gram-positive bacterium.

Iron-sulfur clusters function as cofactors of a wide range of proteins, with diverse molecular roles in both prokaryotic and eukaryotic cells. Dedicated machineries assemble the clusters and deliver them to the final acceptor molecules in a tightly regulated process. In the prototypical Gram-negative bacterium Escherichia coli, the two existing iron-sulfur cluster assembly systems, iron-sulfur cluster (ISC) and sulfur assimilation (SUF) pathways, are closely interconnected. The ISC pathway regulator, IscR, is a transcription factor of the helix-turn-helix type that can coordinate a [2Fe-2S] cluster. Redox conditions and iron or sulfur availability modulate the ligation status of the labile IscR cluster, which in turn determines a switch in DNA sequence specificity of the regulator: cluster-containing IscR can bind to a family of gene promoters (type-1) whereas the clusterless form recognizes only a second group of sequences (type-2). However, iron-sulfur cluster biogenesis in Gram-positive bacteria is not so well characterized, and most organisms of this group display only one of the iron-sulfur cluster assembly systems. A notable exception is the unique Gram-positive dissimilatory metal reducing bacterium Thermincola potens, where genes from both systems could be identified, albeit with a diverging organization from that of Gram-negative bacteria. We demonstrated that one of these genes encodes a functional IscR homolog and is likely involved in the regulation of iron-sulfur cluster biogenesis in T. potens. Structural and biochemical characterization of T. potens and E. coli IscR revealed a strikingly similar architecture and unveiled an unforeseen conservation of the unique mechanism of sequence discrimination characteristic of this distinctive group of transcription regulators.

Prevention and reversal of severe mitochondrial cardiomyopathy by gene therapy in a mouse model of Friedreich's ataxia

Cardiac failure is the most common cause of mortality in Friedreich's ataxia (FRDA), a mitochondrial disease characterized by neurodegeneration, hypertrophic cardiomyopathy and diabetes. FRDA is caused by reduced levels of frataxin (FXN), an essential mitochondrial protein involved in the biosynthesis of iron-sulfur (Fe-S) clusters. Impaired mitochondrial oxidative phosphorylation, bioenergetics imbalance, deficit of Fe-S cluster enzymes and mitochondrial iron overload occur in the myocardium of individuals with FRDA. No treatment exists as yet for FRDA cardiomyopathy. A conditional mouse model with complete frataxin deletion in cardiac and skeletal muscle (Mck-Cre-Fxn(L3/L-) mice) recapitulates most features of FRDA cardiomyopathy, albeit with a more rapid and severe course. Here we show that adeno-associated virus rh10 vector expressing human FXN injected intravenously in these mice fully prevented the onset of cardiac disease. Moreover, later administration of the frataxin-expressing vector, after the onset of heart failure, was able to completely reverse the cardiomyopathy of these mice at the functional, cellular and molecular levels within a few days. Our results demonstrate that cardiomyocytes with severe energy failure and ultrastructure disorganization can be rapidly rescued and remodeled by gene therapy and establish the preclinical proof of concept for the potential of gene therapy in treating FRDA cardiomyopathy.

An Ipomoea batatas iron-sulfur cluster scaffold protein gene, IbNFU1, is involved in salt tolerance.

Iron-sulfur cluster biosynthesis involving the nitrogen fixation (Nif) proteins has been proposed as a general mechanism acting in various organisms. NifU-like protein may play an important role in protecting plants against abiotic and biotic stresses. An iron-sulfur cluster scaffold protein gene, IbNFU1, was isolated from a salt-tolerant sweetpotato (Ipomoea batatas (L.) Lam.) line LM79 in our previous study, but its role in sweetpotato stress tolerance was not investigated. In the present study, the IbNFU1 gene was introduced into a salt-sensitive sweetpotato cv. Lizixiang to characterize its function in salt tolerance. The IbNFU1-overexpressing sweetpotato plants exhibited significantly higher salt tolerance compared with the wild-type. Proline and reduced ascorbate content were significantly increased, whereas malonaldehyde (MDA) content was significantly decreased in the transgenic plants. The activities of superoxide dismutase (SOD) and photosynthesis were significantly enhanced in the transgenic plants. H2O2 was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbNFU1 up-regulated pyrroline-5-carboxylate synthase (P5CS) and pyrroline-5-carboxylate reductase (P5CR) genes under salt stress. The systemic up-regulation of reactive oxygen species (ROS) scavenging genes was found in the transgenic plants under salt stress. These findings suggest that IbNFU1gene is involved in sweetpotato salt tolerance and enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and activating ROS scavenging system.

Physical and Functional Interactions of the E. coli Monothiol Glutaredoxin GrxD Suggest a Role in FeS Apoprotein Maturation (LB132)

The biosynthesis of iron sulfur (FeS) clusters is a highly coordinated process enabled by multi‐protein systems such as Isc and Suf in E. coli. Additional accessory factors including the monothiol glutaredoxin, GrxD, are located outside of these defined systems. GrxD has been proposed to function as a shuttle protein acting to transfer FeS clusters between scaffold and carrier proteins, although specific functional role remain unknown. We have previously linked GrxD in FeS client protein interactions with the radical SAM enzyme MiaB. To address these different hypotheses, we conducted an extensive survey for physical interaction of GrxD with a comprehensive set of FeS cluster biosynthesis factors including cysteine desulfurase components (IscS, SufSE), FeS scaffold (IscU, SufBC2D) , carrier proteins (IscA SufA, ErpA, NfuA), HscA, HscB and various FeS apoproteins. We have employed various methods, including affinity co‐purification, analytical gel filtration and surface plasmon resonance (SPR) to obtain interaction data. To date we have observed no physical interaction between GrxD and any FeS biosynthesis factor except the SufBC2D scaffold and the SufA carrier protein. With SufA a reproducible but extremely weak association is observed by SPR alone (KD >> 700 µM). In contrast we have observed specific interactions between GrxD and two radical SAM enzymes, MiaB (KD =12 µM) and BioB (KD =34 µM). These data strongly indicate a role for GrxD in FeS protein maturation or repair, working downstream of the Suf systemGrant Funding Source: NIH award GM088196 to G.B

Novel aspects of iron sulfur cluster biosynthesis in sulfate reducing bacteria (768.17)

Iron sulfur (FeS) cluster containing proteins are involved in processes targeted by environmentally relevant stressors. These stress conditions, for sulfate reducing bacteria (SRBs) such as Desulfovibro vulgaris Hildenborough (DvH) and Desulfovibrio alaskensis G20, would include exposure to oxygen (and oxygen radicals), the presence of N‐oxyanions such as nitrate and nitrite, and exposure to potentially toxic metals such as selenium, cobalt and chromium. Significantly, FeS cluster damage in key enzymes such as pyruvate‐ferredoxin oxidoreductase (PFOR) has been proposed as the source of inviability of SRBs and other anaerobes when encountering oxygen. Some FeS cluster biosynthesis key factors have been identified in these SRBs by sequence homology. We are characterizing the effect of these mutations on key FeS dependent marker enzymes, including PFOR, formate dehydrogenase and dissimilatory sulfite reductase, in order to determine relative contributions of these factors to FeS cluster biosynthesis. Concurrently, we have also performed fitness profiling of a comprehensive G20 mutant pool under stress conditions known to damage FeS enzymes. We have observed mild fitness defects for the G20 Dde_1444/1445 (sufBC) mutant strains, consistent with reduced marker FeS enzyme activities of the DvH sufB mutant. Interestingly, fitness data has indicated that the sole cysteine desulfurase mutant in G20 (nifS; Dde_3078) displays pleotropic defects, but remains viable, contradicting current dogma regarding FeS cluster biosynthesis.Grant Funding Source: Supported by the U. S. Department of Energy under Contract No. DE‐AC02‐05CH11231

Iron Regulatory Protein-1 Protects against Mitoferrin-1-deficient Porphyria

Mitochondrial iron is essential for the biosynthesis of heme and iron-sulfur ([Fe-S]) clusters in mammalian cells. In developing erythrocytes, iron is imported into the mitochondria by MFRN1 (mitoferrin-1, SLC25A37). Although loss of MFRN1 in zebrafish and mice leads to profound anemia, mutant animals showed no overt signs of porphyria, suggesting that mitochondrial iron deficiency does not result in an accumulation of protoporphyrins. Here, we developed a gene trap model to provide in vitro and in vivo evidence that iron regulatory protein-1 (IRP1) inhibits protoporphyrin accumulation. Mfrn1(+/gt);Irp1(-/-) erythroid cells exhibit a significant increase in protoporphyrin levels. IRP1 attenuates protoporphyrin biosynthesis by binding to the 5'-iron response element (IRE) of alas2 mRNA, inhibiting its translation. Ectopic expression of alas2 harboring a mutant IRE, preventing IRP1 binding, in Mfrn1(gt/gt) cells mimics Irp1 deficiency. Together, our data support a model whereby impaired mitochondrial [Fe-S] cluster biogenesis in Mfrn1(gt/gt) cells results in elevated IRP1 RNA-binding that attenuates ALAS2 mRNA translation and protoporphyrin accumulation.

Mitochondrial iron–sulfur protein biogenesis and human disease

Work during the past 14 years has shown that mitochondria are the primary site for the biosynthesis of iron-sulfur (Fe/S) clusters. In fact, it is this process that renders mitochondria essential for viability of virtually all eukaryotes, because they participate in the synthesis of the Fe/S clusters of key nuclear and cytosolic proteins such as DNA polymerases, DNA helicases, and ABCE1 (Rli1), an ATPase involved in protein synthesis. As a consequence, mitochondrial function is crucial for nuclear DNA synthesis and repair, ribosomal protein synthesis, and numerous other extra-mitochondrial pathways including nucleotide metabolism and cellular iron regulation. Within mitochondria, the synthesis of Fe/S clusters and their insertion into apoproteins is assisted by 17 proteins forming the ISC (iron-sulfur cluster) assembly machinery. Biogenesis of mitochondrial Fe/S proteins can be dissected into three main steps: First, a Fe/S cluster is generated de novo on a scaffold protein. Second, the Fe/S cluster is dislocated from the scaffold and transiently bound to transfer proteins. Third, the latter components, together with specific ISC targeting factors insert the Fe/S cluster into client apoproteins. Disturbances of the first two steps impair the maturation of extra-mitochondrial Fe/S proteins and affect cellular and systemic iron homeostasis. In line with the essential function of mitochondria, genetic mutations in a number of ISC genes lead to severe neurological, hematological and metabolic diseases, often with a fatal outcome in early childhood. In this review we briefly summarize our current functional knowledge on the ISC assembly machinery, and we present a comprehensive overview of the various Fe/S protein assembly diseases.

Distinct Roles of the Salmonella enterica Serovar Typhimurium CyaY and YggX Proteins in the Biosynthesis and Repair of Iron-Sulfur Clusters

Labile [4Fe-4S](2+) clusters found at the active sites of many dehydratases are susceptible to damage by univalent oxidants that convert the clusters to an inactive [3Fe-4S](1+) form. Bacteria repair damaged clusters in a process that does not require de novo protein synthesis or the Isc and Suf cluster assembly pathways. The current study investigates the participation of the bacterial frataxin ortholog CyaY and the YggX protein, which are proposed to play roles in iron trafficking and iron-sulfur cluster repair. Previous reports found that individual mutations in cyaY or yggX were not associated with phenotypic changes in Escherichia coli and Salmonella enterica serovar Typhimurium, suggesting that CyaY and YggX might have functionally redundant roles. However, we have found that individual mutations in cyaY or yggX confer enhanced susceptibility to hydrogen peroxide in Salmonella enterica serovar Typhimurium. In addition, inactivation of the stm3944 open reading frame, which is located immediately upstream of cyaY and which encodes a putative inner membrane protein, dramatically enhances the hydrogen peroxide sensitivity of a cyaY mutant. Overexpression of STM3944 reduces the elevated intracellular free iron levels observed in an S. Typhimurium fur mutant and also reduces the total cellular iron content under conditions of iron overload, suggesting that the stm3944-encoded protein may mediate iron efflux. Mutations in cyaY and yggX have different effects on the activities of the iron-sulfur cluster-containing aconitase, serine deaminase, and NADH dehydrogenase I enzymes of S. Typhimurium under basal conditions or following recovery from oxidative stress. In addition, cyaY and yggX mutations have additive effects on 6-phosphogluconate dehydratase-dependent growth during nitrosative stress, and a cyaY mutation reduces Salmonella virulence in mice. Collectively, these results indicate that CyaY and YggX play distinct supporting roles in iron-sulfur cluster biosynthesis and the repair of labile clusters damaged by univalent oxidants. Salmonella experiences oxidative and nitrosative stress within host phagocytes, and CyaY-dependent maintenance of labile iron-sulfur clusters appears to be important for Salmonella virulence.

Modeling of Friedreich ataxia-related iron overloading cardiomyopathy using patient-specific-induced pluripotent stem cells

Friedreich ataxia (FRDA), a recessive neurodegenerative disorder commonly associated with hypertrophic cardiomyopathy, is due to GAA repeat expansions within the first intron of the frataxin (FXN) gene encoding the mitochondrial protein involved in iron-sulfur cluster biosynthesis. The triplet codon repeats lead to heterochromatin-mediated gene silencing and loss of frataxin. Nevertheless, inadequacy of existing FRDA-cardiac cellular models limited cardiomyopathy studies. We tested the hypothesis that iron homeostasis deregulation accelerates reduction in energy synthesis dynamics which contributes to impaired cardiac calcium homeostasis and contractile force. Silencing of FXN expressions occurred both in somatic FRDA-skin fibroblasts and two of the induced pluripotent stem cells (iPSC) clones; a sign of stress condition was shown in FRDA-iPSC cardiomyocytes with disorganized mitochondrial network and mitochondrial DNA (mtDNA) depletion; hypertrophic cardiac stress responses were observed by an increase in α-actinin-positive cell sizes revealed by FACS analysis as well as elevation in brain natriuretic peptide (BNP) gene expression; the intracellular iron accumulated in FRDA cardiomyocytes might be due to attenuated negative feedback response of transferring receptor (TSFR) expression and positive feedback response of ferritin (FTH1); energy synthesis dynamics, in terms of ATP production rate, was impaired in FRDA-iPSC cardiomyocytes, which were prone to iron overload condition. Energetic insufficiency determined slower Ca(2+) transients by retarding calcium reuptake to sarcoplasmic reticulum (SR) and impaired the positive inotropic and chronotropic responses to adrenergic stimulation. Our data showed for the first time that FRDA-iPSCs cardiac derivatives represent promising models to study cardiac stress response due to impaired iron homeostasis condition and mitochondrial damages. The cardiomyopathy phenotype was accelerated in an iron-overloaded condition early in calcium homeostasis aspect.

O16 – 1908 A homozygous mutation in IB57 involved in intramitochondrial iron-sulfur cluster synthesis causes severe encephalopathy and mypathy in two neonates

Background: Combined OXPHOS deficiencies involving complexes I and II have recently been detected in patients with deficient iron-sulfur cluster (ISC) biogenesis. So far, patients were reported with pathogenic mutations in NFU1 and BOLA3 presenting with severe encephalomyopathy at young age. Objective: Two siblings with combined deficiency of complex I and II were investigated for possible defect in ISC. Patients and methods: The siblings presented soon after birth with severe encephalomyopathy and died in the neonatal period. Biochemical investigations showed increased lactate in serum and increased glycine in CSF. Considering the consanguineous descent a search for genes in homozygous regions related to ISC metabolism was performed. Results: Isolating IBA57 as a strong candidate gene, sequencing detected a homozygous mutation (c.941A>C) in the two siblings and a heterozygous carrier status in both parents. Western blotting showed a severe decrease of CRM for the IBA57 protein. The protein amount in the complexes I and II was significantly decreased. Transfection experiments in HeLa cells demonstrated that the mutation was pathogenic and that excessive degradation of the IBA57 protein was responsible for the defective ISC biosynthesis. Conclusion: This is the first report of a pathogenic mutation in IBA57 in human.

His86 from the N-Terminus of Frataxin Coordinates Iron and Is Required for Fe–S Cluster Synthesis

Human frataxin has a vital role in the biosynthesis of iron-sulfur (Fe-S) clusters in mitochondria, and its deficiency causes the neurodegenerative disease Friedreich's ataxia. Proposed functions for frataxin in the Fe-S pathway include iron donation to the Fe-S cluster machinery and regulation of cysteine desulfurase activity to control the rate of Fe-S production, although further molecular detail is required to distinguish these two possibilities. It is well established that frataxin can coordinate iron using glutamate and aspartate side chains on the protein surface; however, in this work we identify a new iron coordinating residue in the N-terminus of human frataxin using complementary spectroscopic and structural approaches. Further, we demonstrate that His86 in this N-terminal region is required for high affinity iron coordination and iron assembly of Fe-S clusters by ISCU as part of the Fe-S cluster biosynthetic complex. If a binding site that includes His86 is important for Fe-S cluster synthesis as part of its chaperone function, this raises the possibility that either iron binding at the acidic surface of frataxin may be spurious or that it is required for protein-protein interactions with the Fe-S biosynthetic quaternary complex. Our data suggest that iron coordination to frataxin may be significant to the Fe-S cluster biosynthesis pathway in mitochondria.

Ferredoxin Competes with Bacterial Frataxin in Binding to the Desulfurase IscS*

The bacterial iron-sulfur cluster (isc) operon is an essential machine that is highly conserved from bacteria to primates and responsible for iron-sulfur cluster biogenesis. Among its components are the genes for the desulfurase IscS that provides sulfur for cluster formation, and a specialized ferredoxin (Fdx) whose role is still unknown. Preliminary evidence suggests that IscS and Fdx interact but nothing is known about the binding site and the role of the interaction. Here, we have characterized the interaction using a combination of biophysical tools and mutagenesis. By modeling the Fdx·IscS complex based on experimental restraints we show that Fdx competes for the binding site of CyaY, the bacterial ortholog of frataxin and sits in a cavity close to the enzyme active site. By in vivo mutagenesis in bacteria we prove the importance of the surface of interaction for cluster formation. Our data provide the first structural insights into the role of Fdx in cluster assembly.

Frataxin: a protein in search for a function

Reduced levels of the protein frataxin cause the neurodegenerative disease Friedreich's ataxia. Pathology is associated with disruption of iron-sulfur cluster biosynthesis, mitochondrial iron overload, and oxidative stress. Frataxin is a highly conserved iron-binding protein present in most organisms. Despite the intense interest generated since the determination of its pathology, identification of the cellular function of frataxin has so far remained elusive. In this review, we revisit the most significant milestones that have led us to our current understanding of frataxin and its functions. The picture that emerges is that frataxin is a crucial element of one of the most essential cellular machines specialized in iron-sulfur cluster biogenesis. Future developments, therefore, can be expected from further advancements in our comprehension of this machine.

Thermodynamic and Structural Analysis of Human NFU Conformational Chemistry

Human NFU has been implicated in the formation of inorganic sulfide required for cellular iron-sulfur cluster biosynthesis. The protein contains a well-structured N-terminal domain and a C-terminal domain with molten globule characteristics that also contains a thioredoxin-like pair of redox active Cys residues that promote persulfide reductase activity. Recent reports have highlighted the existence of structural flexibility in the ISU/IscU-type scaffold proteins that mediate Fe-S cluster assembly, which is also likely to serve an important role in the pathway to Fe-S cluster maturation. We have previously reported similar structural mobility for the C-terminal domain of human NFU, a protein that has been implicated in the production of sulfide for cluster synthesis, while homologous proteins have also been suggested to serve as Fe-S cluster carriers. Herein we quantitatively characterize the structural stability of the two domains of human NFU and in particular the functional C-terminal domain. The results of differential scanning calorimetry and variable temperature circular dichroism (VTCD) studies have been used to analyze the temperature-dependent structural melting profiles of the N- and C-terminal domains, relative to both full-length NFU and an equimolar ratio of the N- and C-terminal domains, and correlated with structural information derived from NMR data. Calorimetry results indicate that the C-terminal NFU domain undergoes a significant structural stabilization following interaction with the N-terminal domain, which resulted in a novel and distinctive transition melting profile (Tm(sec) = 58.1 ± 0.4 °C, ΔHv(sec) = 60.4 ± 5.3 kcal/mol, Tm(ter) = 49.3 ± 0.3 °C, ΔHv(ter) = 71.8 ± 5.8 kcal/mol). VTCD experiments also revealed a secondary structure transition at 59.2 °C in agreement with calorimetry results. The degree of stabilization was found to be more significant in the full-length NFU, as the C-terminal domain transitions were recorded at higher temperatures (Tm(sec) = 63.3 ± 3.4 °C, ΔHv(sec) = 41.8 ± 8.2 kcal/mol). The interactions between the two domains demonstrated the hallmarks of a hydrophobic character, as increased ionic strength decreased the degree of stabilization of the C-terminal domain. An increase of 2% in α-helix content further supports interaction between the two domains, leading to greater secondary structure stabilization. Heteronuclear single-quantum coherence experiments indicate that the C-terminal domain adopts an alternate tertiary conformation following binding to the N-terminal domain. The structural rigidity of the N-terminal domain leads to an alternative conformation of the C-terminal domain, suggesting that such an interaction, although weaker than that of the covalently attached native NFU, is important for the structural chemistry of the native full-length protein. The results also emphasize the likely general importance of such structural flexibility in select proteins mediating metal cofactor biosynthesis.

Physical and Functional Interactions of a Monothiol Glutaredoxin and an Iron Sulfur Cluster Carrier Protein with the Sulfur-donating Radical S-Adenosyl-l-methionine Enzyme MiaB

The biosynthesis of iron sulfur (FeS) clusters, their trafficking from initial assembly on scaffold proteins via carrier proteins to final incorporation into FeS apoproteins, is a highly coordinated process enabled by multiprotein systems encoded in iscRSUAhscBAfdx and sufABCDSE operons in Escherichia coli. Although these systems are believed to encode all factors required for initial cluster assembly and transfer to FeS carrier proteins, accessory factors such as monothiol glutaredoxin, GrxD, and the FeS carrier protein NfuA are located outside of these defined systems. These factors have been suggested to function both as shuttle proteins acting to transfer clusters between scaffold and carrier proteins and in the final stages of FeS protein assembly by transferring clusters to client FeS apoproteins. Here we implicate both of these factors in client protein interactions. We demonstrate specific interactions between GrxD, NfuA, and the methylthiolase MiaB, a radical S-adenosyl-L-methionine-dependent enzyme involved in the maturation of a subset of tRNAs. We show that GrxD and NfuA physically interact with MiaB with affinities compatible with an in vivo function. We furthermore demonstrate that NfuA is able to transfer its cluster in vitro to MiaB, whereas GrxD is unable to do so. The relevance of these interactions was demonstrated by linking the activity of MiaB with GrxD and NfuA in vivo. We observe a severe defect in in vivo MiaB activity in cells lacking both GrxD and NfuA, suggesting that these proteins could play complementary roles in maturation and repair of MiaB.

The L-Cysteine Desulfurase NFS1 Is Localized in the Cytosol where it Provides the Sulfur for Molybdenum Cofactor Biosynthesis in Humans

In humans, the L-cysteine desulfurase NFS1 plays a crucial role in the mitochondrial iron-sulfur cluster biosynthesis and in the thiomodification of mitochondrial and cytosolic tRNAs. We have previously demonstrated that purified NFS1 is able to transfer sulfur to the C-terminal domain of MOCS3, a cytosolic protein involved in molybdenum cofactor biosynthesis and tRNA thiolation. However, no direct evidence existed so far for the interaction of NFS1 and MOCS3 in the cytosol of human cells. Here, we present direct data to show the interaction of NFS1 and MOCS3 in the cytosol of human cells using Förster resonance energy transfer and a split-EGFP system. The colocalization of NFS1 and MOCS3 in the cytosol was confirmed by immunodetection of fractionated cells and localization studies using confocal fluorescence microscopy. Purified NFS1 was used to reconstitute the lacking molybdoenzyme activity of the Neurospora crassa nit-1 mutant, giving additional evidence that NFS1 is the sulfur donor for Moco biosynthesis in eukaryotes in general.