Cluster Assembly Research Articles

Iron-regulated assembly of the cytosolic iron–sulfur cluster biogenesis machinery

The cytosolic iron–sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. Although the delivery process is regulated by the availability of iron and oxygen, it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we utilized a targeted proteomics assay for monitoring CIA factors and substrates to characterize the CIA machinery. We find that nucleotide-binding protein 1 (NUBP1/NBP35), cytosolic iron–sulfur assembly component 3 (CIAO3/NARFL), and CIA substrates associate with nucleotide-binding protein 2 (NUBP2/CFD1), a component of the CIA scaffold complex. NUBP2 also weakly associates with the CIA targeting complex (MMS19, CIAO1, and CIAO2B) indicating the possible existence of a higher order complex. Interactions between CIAO3 and the CIA scaffold complex are strengthened upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrate that CIAO3 mutants defective in Fe-S cluster binding fail to integrate into the higher order complexes. However, these mutants exhibit stronger associations with CIA substrates under conditions in which the association with the CIA targeting complex is reduced suggesting that CIAO3 and CIA substrates may associate in complexes independently of the CIA targeting complex. Together, our data suggest that CIA components potentially form a metabolon whose assembly is regulated by environmental cues and requires Fe-S cluster incorporation in CIAO3. These findings provide additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization.

The hydrogenosome of Trichomonas vaginalis.

This review is dedicated to the 50th anniversary of the discovery of hydrogenosomes by Miklós Müller and Donald Lindmark, which we will celebrate the following year. It was a long journey from the first observation of enigmatic rows of granules in trichomonads at the end of the 19th century to their first biochemical characterization in 1973. The key experiments by Müller and Lindmark revealed that the isolated granules contain hydrogen-producing hydrogenase, similar to some anaerobic bacteria-a discovery that gave birth to the field of hydrogenosomes. It is also important to acknowledge the parallel work of the team of Apolena Čerkasovová, Jiří Čerkasov, and Jaroslav Kulda, who demonstrated that these granules, similar to mitochondria, produce ATP. However, the evolutionary origin of hydrogenosomes remained enigmatic until the turn of the millennium, when it was finally accepted that hydrogenosomes and mitochondria evolved from a common ancestor. After a historical introduction, the review provides an overview of hydrogenosome biogenesis, hydrogenosomal protein import, and the relationship between the peculiar structure of membrane translocases and its low inner membrane potential due to the lack of respiratory complexes. Next, it summarizes the current state of knowledge on energy metabolism, the oxygen defense system, and iron/sulfur cluster assembly.

Catching a Walker in the Act-DNA Partitioning by ParA Family of Proteins.

Partitioning the replicated genetic material is a crucial process in the cell cycle program of any life form. In bacteria, many plasmids utilize cytoskeletal proteins that include ParM and TubZ, the ancestors of the eukaryotic actin and tubulin, respectively, to segregate the plasmids into the daughter cells. Another distinct class of cytoskeletal proteins, known as the Walker A type Cytoskeletal ATPases (WACA), is unique to Bacteria and Archaea. ParA, a WACA family protein, is involved in DNA partitioning and is more widespread. A centromere-like sequence parS, in the DNA is bound by ParB, an adaptor protein with CTPase activity to form the segregation complex. The ParA ATPase, interacts with the segregation complex and partitions the DNA into the daughter cells. Furthermore, the Walker A motif-containing ParA superfamily of proteins is associated with a diverse set of functions ranging from DNA segregation to cell division, cell polarity, chemotaxis cluster assembly, cellulose biosynthesis and carboxysome maintenance. Unifying principles underlying the varied range of cellular roles in which the ParA superfamily of proteins function are outlined. Here, we provide an overview of the recent findings on the structure and function of the ParB adaptor protein and review the current models and mechanisms by which the ParA family of proteins function in the partitioning of the replicated DNA into the newly born daughter cells.

Protein interactions in the biological assembly of iron-sulfur clusters in Escherichia coli: Molecular and mechanistic aspects of the earliest assembly steps.

This contribution focuses on the earliest steps of the assembly of FeS clusters and their insertion into acceptor apoproteins, that call for transient formation of a 2Fe2S cluster on a scaffold protein from sulfide and iron salts. For the sake of simplicity, this report is essentially limited to the Escherichia coli isc‐encoded proteins and does not take into account agents that modulate the enzymatic synthesis of sulfide by protein in the same operon or the redox events associated with both sulfide generation and conversion of 2Fe2S structures in clusters of higher nuclearity. Therefore, the results discussed here are based on chemical reconstitution systems using inorganic sulfide, ferric salts, and excess thiols. This simplification offers the possibility to address some mechanistic issues related to the role of protein/protein interaction as for modulating: (a) the rate of cluster assembly on scaffold proteins; (b) the stability of the cluster on the scaffold protein; and (c) the rate of transfer to acceptor apoproteins as also influenced by the acceptor concentration. The emerging picture highlights the mechanistic versatility of the systems, that is discussed in terms of the capability of such an apparently simple combination of proteins to cope with various physiological situation. The hypothetical mechanism presented here may represent an additional way of modulating the rate and outcome of the overall process while avoiding potential toxicity issues.

Heterologous expression of ISU1 gene from Fragaria vesca enhances plant tolerance to Fe depletion in Arabidopsis

Iron-sulfur (Fe-S) cluster assembly genes play important roles in plant growth and development. However, their biological function in fruit crops is still unknown, especially in strawberry. In this study, Fe depletion significantly inhibited the growth, photosynthesis, Fe accumulation level and the enzyme activity of Fe-S proteins of aconitase (ACO), nitrate reductase (NiR) and succinate dehydrogenase (SDH) in strawberry seedlings. In addition, 40 Fe-S cluster assembly genes were isolated from strawberry, which were significantly varied among different tissues/organs and were differentially responded to Fe depletion in different tissue parts. In total, 79% of the responsive genes were up-regulated in shoots, while 65% of the responsive genes were down-regulated in roots under Fe depletion. Moreover, the expression level of ISU1 was the highest in strawberry tissues, especially in young fruits, and over-expression of ISU1 gene in Arabidopsis significantly enhanced the Fe accumulation, leaf total chlorophyll, ACO and SDH activities in transgenic lines, and strengthened plant tolerance to Fe depletion. This study provides gene resources to elucidate the molecular mechanisms of Fe-S cluster assembly in strawberry, and lays a theoretical foundation to reveal Fe nutrition and metabolism in Rosaceae fruits.

Free-Energy Landscape and Isomerization Rates of Au4 Clusters at Finite Temperatures.

In metallic nanoparticles, the geometry of atomic positions controls the particle's electronic band structure, polarizability, and catalytic properties. Analyzing the structural properties is a complex problem; the structure of an assembled cluster changes from moment to moment due to thermal fluctuations. Conventional structural analyses based on spectroscopy or diffraction cannot determine the instantaneous structure exactly and can merely provide an averaged structure. Molecular simulations offer an opportunity to examine the assembly and evolution of metallic clusters, as the preferred assemblies and conformations can easily be visualized and explored. Here, we utilize the adaptive biasing force algorithm applied to first-principles molecular dynamics to demonstrate the exploration of a relatively simple system, which permits a comprehensive study of the small metal cluster Au4 in both neutral and charged configurations. Our simulation work offers a quantitative understanding of these clusters' dynamic structure, which is significant for single-site catalytic reactions on metal clusters and provides a starting point for a detailed quantitative understanding of more complex pure metal and alloy clusters' dynamic properties.

Accessing 14-Connected Nets: Continuous Breathing, Hydrophobic Rare-Earth Metal Organic Frameworks Based on 14-c Hexanuclear Clusters with High Affinity for Non-Polar Vapors.

Highly connected metal organic frameworks (MOFs) in which at least one building block has connectivity higher than twelve are very rare and much desirable. We report here the first examples of isostructural 14-connected MOFs, RE-frt-MOF-1, constructed from the assembly of 14-c hexanuclear rare-earth clusters, [RE6(μ3-X)8(COO)12]2- (RE: Y3+, Tb3+, Dy3+, Ho3+, Er3+, Yb3+ and X: OH-/F-) with a tritopic carboxylate-based organic linker. This linker serves as a 3-c and 4-c organic node resulting in the formation of a unique, trinodal (3,4,14)-c framework. RE-frt-MOF-1 are stable in air and alkaline aqueous solutions and show an intriguingly continuous, reversible breathing behavior, between a wide and a narrow-pore phase, upon guest removal. Crystallinity is retained during breathing, and single-crystal X-ray diffraction shed light into the associated structural transformation. Vapor sorption studies performed on Y-frt-MOF-1 revealed a high affinity for non-polar vapors such as n-hexane, cyclohexane, and benzene, displaying type I isotherms with high uptake at low relative pressures (<10-3 p/p0), associated with the hydrophobic nature of the 1D channels and also with their rhombic shape. In contrast, polar vapors such as acetonitrile and ethanol show type V isotherms due to favorable vapor-vapor interactions. Notably these vapors, except cyclohexane, trigger the transition from the narrow to the wide pore phase, accompanied by a remarkable increase in uptake, reaching 70.6, 109, 100.4, and 87.7% for n-hexane, benzene, acetonitrile, and ethanol, respectively.

Sodium-ion-induced reversal of chiral preference observed in proline clusters in the gas phase

Enantioselective self-assembly can be observed in some amino acid cluster ions in the gas phase. Some interesting characters have been previously observed for proline, such as the formation of homochiral dodecamers and chiral preference oscillations of small-sized clusters. However, how alkali metal ions affect the formation and stability of the related homochiral and heterochiral proline clusters is unknown. Herein, the subtle effects of sodium ions on the chiral preference of proline clusters were studied in the gas phase. For the clusters of [Pro10,11+2H]2+ with strong heterochiral preference, the substitution of one proton with one sodium ion reverses their chiral preference, making the ions of [Pro10,11+H + Na]2+ characterized by the homochiral preference. However, the second substitution of sodium ion can reverse the preference once again. The reversal of chiral preference was found to be both cluster-size and alkali metal ion sensitive. Since sodium ions are involved in a wide variety of biological processes and usually inevitably exist in laboratory experiments, the results may have an important impact on the assembly of large-sized proline-related clusters.

Magnetic field effects on the structure and molecular behavior of pigeon iron-sulfur protein.

Pigeon iron-sulfur (Fe-S) cluster assembly 1 homolog (clISCA1) is a target protein for research into the biomagnetoreception mechanism, as the clCRY4/clISCA1 oligomer, a complex composed of the columnar clISCA1 oligomer and the magnetosensor candidate protein cryptochrome-4 (clCRY4) oligomer, tends to orient itself along weak magnetic fields, such as geomagnetic fields, under blue light. To obtain insight into the magnetic orientation mechanism of the clCRY4/clISCA1 oligomer, we inspected magnetic field effects on the structure and molecular behavior of clISCA1 by small angle X-ray scattering analysis. The results indicated that the clISCA1 protomer took the Fe-S cluster-bound globular form and unbound rod-like form. The globular clISCA1 protomer assembled to form columnar oligomers, which allowed for the binding of many Fe-S clusters at the interface between clISCA1 protomers. Moreover, the translational diffusion and the columnar oligomerization of clISCA1 were controlled by the external static magnetic field and Fe-S clusters bound to clISCA1. However, the columnar clISCA1 oligomer was not oriented along the external static magnetic field (~1T) when clCRY4 was not bound to clISCA1. This result indicated that clCRY4 has a function to enhance the magnetic orientational property of clCRY4/clISCA1 oligomer.

Signaling by the integrated stress response kinase PKR is fine-tuned by dynamic clustering.

The double-stranded RNA sensor kinase PKR is one of four integrated stress response (ISR) sensor kinases that phosphorylate the α subunit of eukaryotic initiation factor 2 (eIF2α) in response to stress. The current model of PKR activation considers the formation of back-to-back PKR dimers as a prerequisite for signal propagation. Here we show that PKR signaling involves the assembly of dynamic PKR clusters. PKR clustering is driven by ligand binding to PKR's sensor domain and by front-to-front interfaces between PKR's kinase domains. PKR clusters are discrete, heterogeneous, autonomous coalescences that share some protein components with processing bodies. Strikingly, eIF2α is not recruited to PKR clusters, and PKR cluster disruption enhances eIF2α phosphorylation. Together, these results support a model in which PKR clustering may limit encounters between PKR and eIF2α to buffer downstream signaling and prevent the ISR from misfiring.

Cryo‐EM Structural Determination of Met18, a Scaffold Protein for Iron‐Sulfur Protein Maturation

Iron‐sulfur ([Fe‐S]) clusters are essential cofactors for sustaining life. Because [Fe‐S] clusters play a role in a wide range of cellular processes, defects in their biosynthesis, trafficking, and incorporation into target proteins lead to human diseases. The molecular details of how iron‐sulfur clusters are assembled and transferred to their target proteins are not well understood. Therefore, the goal of this work is to structurally and biophysically characterize proteins involved in the Cytosolic Iron‐Sulfur Cluster Assembly (CIA) pathway. One of these proteins, Met18, is in the CIA targeting complex, and this complex is believed to receive a mature [4Fe‐4S] cluster and transfer it to target proteins downstream. To better understand its function, we determined a 3.3 Å resolution cryo‐EM structure of Met18. In the absence of other CIA targeting complex proteins and target proteins, we observed that Met18 forms a hexamer with its N‐terminus exposed and its C‐terminus buried. Conserved sequences within Met18 assist in forming the hexamer. Upon the addition of Cia2, a CIA targeting complex protein, the hexamer appears to be broken up. We hypothesize that the hexamer is a storage form of Met18 that protects itself from ubiquitination and proteosomal degradation. Here we present the cryo‐EM structure determination of the Met18 hexamer.

Convergent gene clusters underpin hyperforin biosynthesis in St John's wort.

The meroterpenoid hyperforin is responsible for the antidepressant activity of St John's wort extracts, but the genes controlling its biosynthesis are unknown. Using genome mining and biochemical work, we characterize two biosynthetic gene clusters (BGCs) that encode the first three steps in the biosynthesis of hyperforin precursors. The findings of syntenic and phylogenetic analyses reveal the parallel assembly of the two BGCs. The syntenous BGC in Mesua ferrea indicates that the first cluster was assembled before the divergence of the Hypericaceae and Calophyllaceae families. The assembly of the second cluster is the result of a coalescence of genomic fragments after a major duplication event. The differences between the two BGCs - in terms of gene expression, response to methyl jasmonate, substrate specificity and subcellular localization of key enzymes - suggest that the presence of the two clusters could serve to generate separate pools of precursors. The parallel assembly of two BGCs with similar compositions in a single plant species is uncommon, and our work provides insights into how and when these gene clusters form. Our discovery helps to advance our understanding of the evolution of plant specialized metabolism and its genomic organization. Additionally, our results offer a foundation from which hyperforin biosynthesis can be more fully understood, and which can be used in future metabolic engineering applications.

The nano- and meso-scale structure of amorphous calcium carbonate

Understanding the underlying processes of biomineralization is crucial to a range of disciplines allowing us to quantify the effects of climate change on marine organisms, decipher the details of paleoclimate records and advance the development of biomimetic materials. Many biological minerals form via intermediate amorphous phases, which are hard to characterize due to their transient nature and a lack of long-range order. Here, using Monte Carlo simulations constrained by X-ray and neutron scattering data together with model building, we demonstrate a method for determining the structure of these intermediates with a study of amorphous calcium carbonate (ACC) which is a precursor in the bio-formation of crystalline calcium carbonates. We find that ACC consists of highly ordered anhydrous nano-domains of approx. 2 nm that can be described as nanocrystalline. These nano-domains are held together by an interstitial net-like matrix of water molecules which generate, on the mesoscale, a heterogeneous and gel-like structure of ACC. We probed the structural stability and dynamics of our model on the nanosecond timescale by molecular dynamics simulations. These simulations revealed a gel-like and glassy nature of ACC due to the water molecules and carbonate ions in the interstitial matrix featuring pronounced orientational and translational flexibility. This allows for viscous mobility with diffusion constants four to five orders of magnitude lower than those observed in solutions. Small and ultra-small angle neutron scattering indicates a hierarchically-ordered organization of ACC across length scales that allow us, based on our nano-domain model, to build a comprehensive picture of ACC formation by cluster assembly from solution. This contribution provides a new atomic-scale understanding of ACC and provides a framework for the general exploration of biomineralization and biomimetic processes.

Exposure to 3,3',4,4',5-Pentachlorobiphenyl (PCB126) Causes Widespread DNA Hypomethylation in Adult Zebrafish Testis.

Exposure to environmental toxicants during preconception has been shown to affect offspring health and epigenetic mechanisms such as DNA methylation are hypothesized to be involved in adverse outcomes. However, studies addressing the effects of exposure to environmental toxicants during preconception on epigenetic changes in gametes are limited. The objective of this study is to determine the effect of preconceptional exposure to a dioxin-like polychlorinated biphenyl (3,3',4,4',5-pentachlorobiphenyl [PCB126]) on DNA methylation and gene expression in testis. Adult zebrafish were exposed to 3 and 10 nM PCB126 for 24 h and testis tissue was sampled at 7 days postexposure for histology, DNA methylation, and gene expression profiling. Reduced representation bisulfite sequencing revealed 37 and 92 differentially methylated regions (DMRs) in response to 3 and 10 nM PCB126 exposures, respectively. Among them, 19 DMRs were found to be common between both PCB126 treatment groups. Gene ontology (GO) analysis of DMRs revealed that enrichment of terms such as RNA processing, iron-sulfur cluster assembly, and gluconeogenesis. Gene expression profiling showed differential expression of 40 and 1621 genes in response to 3 and 10 nM PCB126 exposures, respectively. GO analysis of differentially expressed genes revealed enrichment of terms related to xenobiotic metabolism, oxidative stress, and immune function. There is no overlap in the GO terms or individual genes between DNA methylation and RNA sequencing results, but functionally many of the altered pathways have been shown to cause spermatogenic defects.

ITRAQ Proteomic Analysis of Wheat (Triticum aestivum L.) Genotypes Differing in Waterlogging Tolerance.

Transient and chronic waterlogging constrains crop production in many regions of the world. Here, we invoke a novel iTRAQ-based proteomic strategy to elicit protein synthesis and regulation responses to waterlogging in tolerant (XM 55) and sensitive genotypes (YM 158). Of the 7,710 proteins identified, 16 were distinct between the two genotypes under waterlogging, partially defining a proteomic basis for waterlogging tolerance (and sensitivity). We found that 11 proteins were up-regulated and 5 proteins were down-regulated; the former included an Fe-S cluster assembly factor, heat shock cognate 70, GTP-binding protein SAR1A-like and CBS domain-containing protein. Down-regulated proteins contained photosystem II reaction center protein H, carotenoid 9, 10 (9′, 10′)-cleavage dioxygenase-like, psbP-like protein 1 and mitochondrial ATPase inhibitor. We showed that nine proteins responded to waterlogging with non-cultivar specificity: these included 3-isopropylmalate dehydratase large subunit, solanesyl-diphosphate synthase 2, DEAD-box ATP-dependent RNA helicase 3, and 3 predicted or uncharacterized proteins. Sixteen of the 28 selected proteins showed consistent expression patterns between mRNA and protein levels. We conclude that waterlogging stress may redirect protein synthesis, reduce chlorophyll synthesis and enzyme abundance involved in photorespiration, thus influencing synthesis of other metabolic enzymes. Collectively, these factors accelerate the accumulation of harmful metabolites in leaves in waterlogging-susceptible genotypes. The differentially expressed proteins enumerated here could be used as biological markers for enhancing waterlogging tolerance as part of future crop breeding programs.

Structural and Functional Analysis of DndE Involved in DNA Phosphorothioation in the Haloalkaliphilic Archaea Natronorubrum bangense JCM10635.

ABSTRACTPhosphorothioate (PT) modification, a sequence-specific modification that replaces the nonbridging oxygen atom with sulfur in a DNA phosphodiester through the gene products of dndABCDE or sspABCD, is widely distributed in prokaryotes. DNA PT modification functions together with gene products encoded by dndFGH, pbeABCD, or sspE to form defense systems that can protect against invasion by exogenous DNA particles. While the functions of the multiple enzymes in the PT system have been elucidated, the exact role of DndE in the PT process is still obscure. Here, we solved the crystal structure of DndE from the haloalkaliphilic archaeal strain Natronorubrum bangense JCM10635 at a resolution of 2.31 Å. Unlike the tetrameric conformation of DndE in Escherichia coli B7A, DndE from N. bangense JCM10635 exists in a monomeric conformation and can catalyze the conversion of supercoiled DNA to nicked or linearized products. Moreover, DndE exhibits preferential binding affinity to nicked DNA by virtue of the R19- and K23-containing positively charged surface. This work provides insight into how DndE functions in PT modification and the potential sulfur incorporation mechanism of DNA PT modification.

Yeast cells depleted of the frataxin homolog Yfh1 redistribute cellular iron: Studies using Mössbauer spectroscopy and mathematical modeling

The neurodegenerative disease Friedreich’s ataxia arises from a deficiency of frataxin, a protein that promotes iron–sulfur cluster (ISC) assembly in mitochondria. Here, primarily using Mössbauer spectroscopy, we investigated the iron content of a yeast strain in which expression of yeast frataxin homolog 1 (Yfh1), oxygenation conditions, iron concentrations, and metabolic modes were varied. We found that aerobic fermenting Yfh1-depleted cells grew slowly and accumulated FeIII nanoparticles, unlike WT cells. Under hypoxic conditions, the same mutant cells grew at rates similar to WT cells, had similar iron content, and were dominated by FeII rather than FeIII nanoparticles. Furthermore, mitochondria from mutant hypoxic cells contained approximately the same levels of ISCs as WT cells, confirming that Yfh1 is not required for ISC assembly. These cells also did not accumulate excessive iron, indicating that iron accumulation into yfh1-deficient mitochondria is stimulated by O2. In addition, in aerobic WT cells, we found that vacuoles stored FeIII, whereas under hypoxic fermenting conditions, vacuolar iron was reduced to FeII. Under respiring conditions, vacuoles of Yfh1-deficient cells contained FeIII, and nanoparticles accumulated only under aerobic conditions. Taken together, these results informed a mathematical model of iron trafficking and regulation in cells that could semiquantitatively simulate the Yfh1-deficiency phenotype. Simulations suggested partially independent regulation in which cellular iron import is regulated by ISC activity in mitochondria, mitochondrial iron import is regulated by a mitochondrial FeII pool, and vacuolar iron import is regulated by cytosolic FeII and mitochondrial ISC activity.

White‐Light Emission and Circularly Polarized Luminescence from a Chiral Copper(I) Coordination Polymer through Symmetry‐Breaking Crystallization

AbstractAchieving white‐light emission, especially white circularly polarized luminescence (CPL) from a single‐phase material is challenging. Herein, a pair of chiral CuI coordination polymers (1‐M and 1‐P) have been prepared by the asymmetrical assembly of achiral ligands and Cu2I2 clusters. The compounds display dual emission bands and can be used as single‐phase white‐light phosphors, achieving a “warm”‐white‐light‐emitting diode with an ultra‐high color rendering index (CRI) of 93.4 and an appropriate correlated color temperature (CCT) of 3632 K. Meanwhile, corresponding CPL signals with maximum dissymmetry factor |glum|=8×10−3 have been observed. Hence, intrinsic white‐light emission and CPL have been realized simultaneously in coordination polymers for the first time. This work gains insight into the nature of chiral assembly from achiral units and offers a prospect for the development of single‐phase white‐CPL materials.

White-Light Emission and Circularly Polarized Luminescence from a Chiral Copper(I) Coordination Polymer through Symmetry-Breaking Crystallization.

Achieving white-light emission, especially white circularly polarized luminescence (CPL) from a single-phase material is challenging. Herein, a pair of chiral CuI coordination polymers (1-M and 1-P) have been prepared by the asymmetrical assembly of achiral ligands and Cu2 I2 clusters. The compounds display dual emission bands and can be used as single-phase white-light phosphors, achieving a "warm"-white-light-emitting diode with an ultra-high color rendering index (CRI) of 93.4 and an appropriate correlated color temperature (CCT) of 3632 K. Meanwhile, corresponding CPL signals with maximum dissymmetry factor |glum |=8×10-3 have been observed. Hence, intrinsic white-light emission and CPL have been realized simultaneously in coordination polymers for the first time. This work gains insight into the nature of chiral assembly from achiral units and offers a prospect for the development of single-phase white-CPL materials.

Protein-Interaction Affinity Gradient Drives [4Fe-4S] Cluster Insertion in Human Lipoyl Synthase.

Human lipoyl synthase (LIAS) is an enzyme containing two [4Fe–4S] clusters (named FeSRS and FeSaux) involved in the biosynthesis of the lipoyl cofactor. The mechanism by which a [4Fe–4S] cluster is inserted into LIAS has thus far remained elusive. Here we show that NFU1 and ISCA1 of the mitochondrial iron–sulfur cluster assembly machinery, via forming a heterodimeric complex, are the key factors for the insertion of a [4Fe–4S] cluster into the FeSRS site of LIAS. In this process, the crucial actor is the C-domain of NFU1, which, by exploiting a protein-interaction affinity gradient increasing from ISCA1 to LIAS, drives the cluster to its final destination.