Cluster Assembly Pathway Research Articles

Genome mining of Mycobacterium tuberculosis: targeting SufD as a novel drug candidate through in silico characterization and inhibitor screening.

Tuberculosis (TB) stands as the second most fatal infectious disease globally, causing 1.3 million deaths in 2022. The resurgence of TB and the alarming rise of antibiotic resistance demand urgent call to develop novel antituberculosis drugs. Despite concerted efforts to control TB, the disease persists and spreads rapidly on a global scale. Targeting stress response pathways in Mycobacterium tuberculosis (Mtb) has become imperative to achieve complete eradication. This study employs subtractive genomics to identify and prioritize potential drug targets among the hypothetical proteins of Mtb, focusing on indispensable pathways. Amongst 177 essential hypothetical proteins, 152 were nonhomologous to human. These proteins participated in 34 pathways, and a 20-fold enrichment of SUF pathway genes led to its selection as a target pathway. Fe-S clusters are fundamental, widely distributed protein cofactors involved in vital cellular processes. The survival of Mtb in a hypoxic environment relies on the iron-sulfur (Fe-S) cluster biogenesis pathway for the repair of damaged Fe-S clusters. It also protects pathogen against drugs, ensuring controlled iron utilization and contributing to drug resistance. In Mtb, six proteins of Fe-S cluster assembly pathway are encoded by the suf operon. The present study was focused on SufD because of its role in iron acquisition and prevention of Fenton reaction. The research further delves into the in silico characterization of SufD, utilizing bioinformatics tools for sequence and structure based analysis. The protein's structural features, including the identification of conserved regions, motifs, and 3D structure prediction enhanced functional annotation. Target based virtual screening of compounds from the ChEMBL database resulted in 12 inhibitors with best binding affinities. Drug likeness and ADMET profiling of potential inhibitors identified promising compounds with favorable drug-like properties. The study also involved cloning in SUMO-pRSF-Duet1 expression vector, overexpression, and purification of recombinant SufD from E. coli BL21 (DE3) cells. Optimization of expression conditions resulted in soluble production, and subsequent purification highlighting the efficacy of the SUMO fusion system for challenging Mtb proteins in E. coli. These findings provide valuable insights into pharmacological targets for future experimental studies, holding promise for the development of targeted therapy against Mtb.

Structural investigations of the cytosolic iron-sulfur cluster assembly pathway late acting proteins

Structural investigations of the cytosolic iron-sulfur cluster assembly pathway late acting proteins

Structural and biochemical investigations of a HEAT-repeat protein involved in the cytosolic iron-sulfur cluster assembly pathway

Iron-sulfur clusters are essential for life and defects in their biosynthesis lead to human diseases. The mechanism of cluster assembly and delivery to cytosolic and nuclear client proteins via the cytosolic iron-sulfur cluster assembly (CIA) pathway is not well understood. Here we report cryo-EM structures of the HEAT-repeat protein Met18 from Saccharomyces cerevisiae, a key component of the CIA targeting complex (CTC) that identifies cytosolic and nuclear client proteins and delivers a mature iron-sulfur cluster. We find that in the absence of other CTC proteins, Met18 adopts tetrameric and hexameric states. Using mass photometry and negative stain EM, we show that upon the addition of Cia2, these higher order oligomeric states of Met18 disassemble. We also use pulldown assays to identify residues of critical importance for Cia2 binding and recognition of the Leu1 client, many of which are buried when Met18 oligomerizes. Our structures show conformations of Met18 that have not been previously observed in any Met18 homolog, lending support to the idea that a highly flexible Met18 may be key to how the CTC is able to deliver iron-sulfur clusters to client proteins of various sizes and shapes, i.e. Met18 conforms to the dimensions needed.

NARFL deficiency caused mitochondrial dysfunction in lung cancer cells by HIF-1α–DNMT1 axis

NARFL was reported to be a component of cytosolic iron–sulfur cluster assembly pathway and a causative gene of the diffused pulmonary arteriovenous malformations (dPAVMs). NARFL knockout dramatically impaired mitochondrial integrity in mice, which might promote mitochondrial dysfunction and lead to worse survival rate of lung cancer. However, the underlying molecular mechanism of NARFL deficiency in non-small cell lung cancer (NSCLC) is unknown. Knockdown assay was performed in A549 and H1299 cells. The protein levels of HIF-1α and DNMT1 were measured, and then Complex I activity, mtDNA copy numbers and mRNA levels of mtND genes were determined. Cisplatin resistance and cell proliferation were conducted using CCK8 assay. Cell migration and invasion were detected using wound heal assay and transwell assay. Survival analysis of lung cancer patients and KM plotter database were used for evaluating the potential value of NARFL deficiency. NARFL protein was expressed in two cell lines and knockdown assay significantly reduced its levels. Knockdown NARFL increased the protein levels of HIF-1α and DNMT1, and downregulated the mRNA levels of ND genes, mitochondrial Complex I activity, mtDNA copy number, and ATP levels. The mitochondrial dysfunction caused by NARFL deficiency were ameliorated by siHIF-1α and DNMT1 inhibitor. Knockdown NARFL increased the drug resistance and cell migration, and siHIF-1α reversed this effect. Moreover, NSCLC patients with NARFL deficiency had a poor survival rate using a tissue array and KM plotter database, and it would be a target for cancer prognosis and treatment. NARFL deficiency caused dysregulation of energy metabolism in lung cancer cells via HIF-1α–DNMT1 axis, which promoted drug resistance and cell migration. It provided a potential target for treatment and prognosis of lung cancer.

Drosophila melanogaster frataxin: protein crystal and predicted solution structure with identification of the iron-binding regions.

Friedreich's ataxia (FRDA) is a hereditary cardiodegenerative and neurodegenerative disease that affects 1 in 50 000 Americans. FRDA arises from either a cellular inability to produce sufficient quantities or the production of a nonfunctional form of the protein frataxin, a key molecule associated with mitochondrial iron-sulfur cluster biosynthesis. Within the mitochondrial iron-sulfur cluster (ISC) assembly pathway, frataxin serves as an allosteric regulator for cysteine desulfurase, the enzyme that provides sulfur for [2Fe-2S] cluster assembly. Frataxin is a known iron-binding protein and is also linked to the delivery of ferrous ions to the scaffold protein, the ISC molecule responsible for the direct assembly of [2Fe-2S] clusters. The goal of this report is to provide structural details of the Drosophila melanogaster frataxin ortholog (Dfh), using both X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, in order to provide the foundational insight needed to understand the structure-function correlation of the protein. Additionally, NMR iron(II) titrations were used to provide metal contacts on the protein to better understand how it binds iron and aids its delivery to the ISC scaffold protein. Here, the structural and functional similarities of Dfh to its orthologs are also outlined. Structural data show that bacterial, yeast, human and Drosophila frataxins are structurally similar, apart from a structured C-terminus in Dfh that is likely to aid in protein stability. The iron-binding location on helix 1 and strand 1 of Dfh is also conserved across orthologs.

The cytosolic iron-sulphur cluster assembly mechanism in grapevine is one target of a virulent Crinkler effector from Plasmopara viticola.

Grapevine downy mildew is one of the most devastating diseases in grape production worldwide, but its pathogenesis remains largely unknown. A thorough understanding of the interaction between grapevine and the causal agent, Plasmopara viticola, is helpful to develop alternative disease control measures. Effector proteins that could be secreted to the interaction interface by pathogens are responsible for the susceptibility of host plants. In this study, a Crinkler effector, named PvCRN17, which is from P.viticola and showed virulent effects towards Nicotiana benthamiana previously, was further investigated. Consistently, PvCRN17 showed a virulent effect on grapevine plants. Protein-protein interaction experiments identified grapevine VAE7L1 (Vitis protein ASYMMETRIC LEAVES 1/2 ENHANCER 7-Like 1) as one target of PvCRN17. VAE7L1 was found to interact with VvCIA1 and VvAE7, thus it may function in the cytosolic iron-sulphur cluster assembly (CIA) pathway. Transient expression of VAE7L1 in Vitis riparia and N.benthamiana leaves enhanced the host resistance to oomycete pathogens. Downstream of the CIA pathway in grapevine, three iron-sulphur (Fe-S) proteins showed an enhancing effect on the disease resistance of N.benthamiana. Competitive co-immunoprecipitation assay showed PvCRN17 could compete with VvCIA1 to bind with VAE7L1 and VvAE7. Moreover, PvCRN17 and VAE7L1 were colocalized at the plasma membrane of the plant cell. To conclude, after intruding into the grapevine cell, PvCRN17 would compete with VCIA1 to bind with VAE7L1 and VAE7, demolishing the CIA Fe-S cluster transfer complex, interrupting the maturation of Fe-S proteins, to suppress Fe-S proteins-mediated defence responses.

Isolation and characterization of IbAE7-like 1 gene encoding the cytosolic iron-sulfur cluster assembly pathway in response to skinning injury in sweetpotato

Abstract. Effendy J. 2022. Isolation and characterization of IbAE7-like 1 gene encoding the cytosolic iron-sulfur cluster assembly pathway in response to skinning injury in sweetpotato. Biodiversitas 23: 4223-4233. Skinning injury is a major constrain in postharvest loss in sweetpotato due to the loss of skin from the surface of the roots. ASYMMETRIC LEAVES1/2 ENHANCER7(AE7) is an important cytosolic iron-sulfur (Fe-S) protein assembly (CIA) pathway. A partial cDNAIbI51 encoding AE7-like 1 was isolated from a skinning injury cDNA library to evaluate its potential role in response to skinning injury. The open reading frameof IbAE7-like 1 consisted of 324 nucleotides and the deduced polypeptide sequences consistedof 108 amino acids with missing 46 amino acid residues at 5? end. The amino acid sequence ofIbAE7-like 1 shared 100% identity toAE7-like 1 previously identified in Ipomoea triloba and Ipomoea nil and 89.91-100% identity from other plants species. The findings indicated that AE7-like 1 was highly conserved in Ipomoea sp. In addition to similarity to AE7-like 1, IbAE7-like 1 also showed similarities to MIP18 (89.91-93.52%); DUF59 (91.67%); FAM96B (90.74%); and proteins with unknown function (90.74-91.67%). IbAE7-like 1 mRNA transcripts showed transient expression in response to skinning injury. The phylogenetic analysis classified IbAE7-like 1 into three main classes. This study revealed IbAE7-like 1 may play a key role in plant defense and wound repair to prevent DNA damage due to skinning injuryin storage roots of sweetpotato.

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.

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.

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.

Pathogenicity Prediction of Potential Variants of Iba57 Gene Causing Heredity Spastic Paraplegia Through Mitochondrial Dysfunction by using Computational Tools

IBA57 is the protein-coding gene that involves in the functional activity of mitochondria. IBA57 protein is related to the iron-sulfur cluster assembly pathway that regulates specific proteins. Mutation in the IBA57 gene damaged the functional activity of mitochondria and respiratory complexes. Heredity spastic paraplegia is a heterogeneous group of neurological disorders that affect the spinal cords and nerves in the human body. In this study, we use bioinformatics tools to analyze the structural and functional information of the IBA57 gene. Advanced in-silico approaches apply to find the pathogenic variants. To find these novel mutations, IBA57 protein data is retrieved through UniProt and gnomAD databases. Pathogenicity of variants was prognosticated through these software (SNP2, PhD-SNPg, and SNPs&go). Furthermore, the strategy applied to these variants is to check out the stability of IBA57 protein, by using DynaMut, CUPSAT, I-Stable, MAESTRO, and YASARA. IBA57 proteins are affected during the splicing mechanism. If mutation gets repeated on the splicing sites then there is a probability that the mutation can disturb the gene sequence as a result the protein formed also gets changed. ScanProsite protein databases and UCSF Chimera (PDB ID: 6QE4) were used to analyze the structural and functional domains of the protein. The deleterious effect of mutations on the structure of IBA57 protein with different parameters and contact/clashes among mutated residues also presented by chimera. These findings conclude that 34 high-risk pathogenic missense variants can cause the change of amino acid properties of IBA57 protein. Hence, this study revealed the importance of bioinformatics algorithms to figure out highly pathogenic variants linked with the structural and functional relationship of IBA57 protein.

The cytosolic iron\u2013sulfur cluster assembly (CIA) pathway is required for replication stress tolerance of cancer cells to Chk1 and ATR inhibitors

The relationship between ATR/Chk1 activity and replication stress, coupled with the development of potent and tolerable inhibitors of this pathway, has led to the clinical exploration of ATR and Chk1 inhibitors (ATRi/Chk1i) as anticancer therapies for single-agent or combinatorial application. The clinical efficacy of these therapies relies on the ability to ascertain which patient populations are most likely to benefit, so there is intense interest in identifying predictive biomarkers of response. To comprehensively evaluate the components that modulate cancer cell sensitivity to replication stress induced by Chk1i, we performed a synthetic-lethal drop-out screen in a cell line derived from a patient with triple-negative breast cancer (TNBC), using a pooled barcoded shRNA library targeting ~350 genes involved in DNA replication, DNA damage repair, and cycle progression. In addition, we sought to compare the relative requirement of these genes when DNA fidelity is challenged by clinically relevant anticancer breast cancer drugs, including cisplatin and PARP1/2 inhibitors, that have different mechanisms of action. This global comparison is critical for understanding not only which agents should be used together for combinatorial therapies in breast cancer patients, but also the genetic context in which these therapies will be most effective, and when a single-agent therapy will be sufficient to provide maximum therapeutic benefit to the patient. We identified unique potentiators of response to ATRi/Chk1i and describe a new role for components of the cytosolic iron–sulfur assembly (CIA) pathway, MMS19 and CIA2B-FAM96B, in replication stress tolerance of TNBC.

Molecular Details of the Frataxin-Scaffold Interaction during Mitochondrial Fe-S Cluster Assembly.

Iron–sulfur clusters are essential to almost every life form and utilized for their unique structural and redox-targeted activities within cells during many cellular pathways. Although there are three different Fe–S cluster assembly pathways in prokaryotes (the NIF, SUF and ISC pathways) and two in eukaryotes (CIA and ISC pathways), the iron–sulfur cluster (ISC) pathway serves as the central mechanism for providing 2Fe–2S clusters, directly and indirectly, throughout the entire cell in eukaryotes. Proteins central to the eukaryotic ISC cluster assembly complex include the cysteine desulfurase, a cysteine desulfurase accessory protein, the acyl carrier protein, the scaffold protein and frataxin (in humans, NFS1, ISD11, ACP, ISCU and FXN, respectively). Recent molecular details of this complex (labeled NIAUF from the first letter from each ISC protein outlined earlier), which exists as a dimeric pentamer, have provided real structural insight into how these partner proteins arrange themselves around the cysteine desulfurase, the core dimer of the (NIAUF)2 complex. In this review, we focus on both frataxin and the scaffold within the human, fly and yeast model systems to provide a better understanding of the biophysical characteristics of each protein alone and within the FXN/ISCU complex as it exists within the larger NIAUF construct. These details support a complex dynamic interaction between the FXN and ISCU proteins when both are part of the NIAUF complex and this provides additional insight into the coordinated mechanism of Fe–S cluster assembly.

Methods to Unravel the Roles of ATPases in Fe-S Cluster Biosynthesis.

Complex biosynthetic pathways are required for the assembly and insertion of iron-sulfur (Fe-S) cluster cofactors. Each of the four cluster biogenesis systems that have been discovered requires at least one ATPase. Generally, the function of nucleotide hydrolysis in Fe-S cluster biogenesis is understudied. For example, the cytosolic Fe-S cluster assembly (CIA) pathway is proposed to begin with a scaffold, which assembles nascent Fe-S clusters destined for cytosolic and nuclear enzymes. This scaffold, comprised of Nbp35 and Cfd1 in yeast, possesses an ATPase site that is necessary for CIA function, but the role of nucleotide hydrolysis is poorly understood. Herein, we describe the in vitro methods that have been developed to uncover how the ATPase site of the scaffold regulates interaction with one of its partner proteins, Dre2. We describe a qualitative affinity copurification assay and a quantitative assay for evaluating the dissociation constant for the scaffold-partner protein complex. Finally, we describe kinetic methods to measure the kcat and KM values for ATP hydrolysis by the scaffold-partner protein complex and the execution of the ATPase assays in an anaerobic environment. These methods could be applied to study other ATPases to advance our mechanistic understanding of nucleotide hydrolases involved in metallocluster biogenesis.

Highly Reduced Plastid Genomes of the Non-photosynthetic Dictyochophyceans Pteridomonas spp. (Ochrophyta, SAR) Are Retained for tRNA-Glu-Based Organellar Heme Biosynthesis.

Organisms that have lost their photosynthetic capabilities are present in a variety of eukaryotic lineages, such as plants and disparate algal groups. Most of such non-photosynthetic eukaryotes still carry plastids, as these organelles retain essential biological functions. Most non-photosynthetic plastids possess genomes with varied protein-coding contents. Such remnant plastids are known to be present in the non-photosynthetic, bacteriovorous alga Pteridomonas danica (Dictyochophyceae, Ochrophyta), which, regardless of its obligatory heterotrophic lifestyle, has been reported to retain the typically plastid-encoded gene for ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) large subunit (rbcL). The presence of rbcL without photosynthetic activity suggests that investigating the function of plastids in Pteridomonas spp. would likely bring unique insights into understanding the reductive evolution of plastids, their genomes, and plastid functions retained after the loss of photosynthesis. In this study, we demonstrate that two newly established strains of the non-photosynthetic genus Pteridomonas possess highly reduced plastid genomes lacking rbcL gene, in contrast to the previous report. Interestingly, we discovered that all plastid-encoded proteins in Pteridomonas spp. are involved only in housekeeping processes (e.g., transcription, translation and protein degradation), indicating that all metabolite synthesis pathways in their plastids are supported fully by nuclear genome-encoded proteins. Moreover, through an in-depth survey of the available transcriptomic data of another strain of the genus, we detected no candidate sequences for nuclear-encoded, plastid-directed Fe–S cluster assembly pathway proteins, suggesting complete loss of this pathway in the organelle, despite its widespread conservation in non-photosynthetic plastids. Instead, the transcriptome contains plastid-targeted components of heme biosynthesis, glycolysis, and pentose phosphate pathways. The retention of the plastid genomes in Pteridomonas spp. is not explained by the Suf-mediated constraint against loss of plastid genomes, previously proposed for Alveolates, as they lack Suf genes. Bearing all these findings in mind, we propose the hypothesis that plastid DNA is retained in Pteridomonas spp. for the purpose of providing glutamyl-tRNA, encoded by trnE gene, as a substrate for the heme biosynthesis pathway.

A pathway for assembling [4Fe-4S]2+ clusters in mitochondrial iron-sulfur protein biogenesis.

During its late steps, the mitochondrial iron-sulfur cluster (ISC) assembly machinery leads to the formation of [4Fe-4S] clusters. Invivo studies revealed that several proteins are implicated in the biosynthesis and trafficking of [4Fe-4S] clusters in mitochondria. However, they do not provide a clear picture into how these proteins cooperate. Here, we showed that three late-acting components of the mitochondrial ISC assembly machinery (GLRX5, BOLA3, and NFU1) are part of a ISC assembly pathway leading to the synthesis of a [4Fe-4S]2+ cluster on NFU1. We showed that the [2Fe-2S]2+ GLRX5-BOLA3 complex transfers its cluster to monomeric apo NFU1 to form, in the presence of a reductant, a [4Fe-4S]2+ cluster bound to dimeric NFU1. The cluster formation on NFU1 does not occur with [2Fe-2S]2+ GLRX5, and thus, the [4Fe-4S] cluster assembly pathway is activated only in the presence of BOLA3. These results define NFU1 as an 'assembler' of [4Fe-4S] clusters, that is, a protein able of converting two [2Fe-2S]2+ clusters into a [4Fe-4S]2+ cluster. Finally, we found that the [4Fe-4S]2+ cluster bound to NFU1 has a coordination site which is easily accessible to sulfur-containing ligands, as is typically observed in metallochaperones. This finding supports a role for NFU1 in promoting rapid and controlled cluster-exchange reaction.

Nubp2 is required for cranial neural crest survival in the mouse

The N-ethyl-N-nitrosourea (ENU) ←forward genetic screen is a useful tool for the unbiased discovery of novel mechanisms regulating developmental processes. We recovered the dorothy mutation in such a screen designed to recover recessive mutations affecting craniofacial development in the mouse. Dorothy embryos die prenatally and exhibit many striking phenotypes commonly associated with ciliopathies, including a severe midfacial clefting phenotype. We used exome sequencing to discover a missense mutation in nucleotide binding protein 2 (Nubp2) to be causative. This finding was confirmed by a complementation assay with the dorothy allele and an independent Nubp2 null allele (Nubp2null). We demonstrated that Nubp2 is indispensable for embryogenesis. NUBP2 is implicated in both the cytosolic iron/sulfur cluster assembly pathway and negative regulation of ciliogenesis. Conditional ablation of Nubp2 in the neural crest lineage with Wnt1-cre recapitulates the dorothy craniofacial phenotype. Using this model, we found that the proportion of ciliated cells in the craniofacial mesenchyme was unchanged, and that markers of the SHH, FGF, and BMP signaling pathways are unaltered. Finally, we show evidence that the phenotype results from a marked increase in apoptosis within the craniofacial mesenchyme.

Backbone resonance assignments and secondary structure of the apo-Drosophila melanogaster frataxin homolog (Dfh).

Friedreich's ataxia, the most prevalent hereditary ataxia, is caused by a patient's inability to produce a viable form of the protein frataxin. Frataxin plays an essential role in cellular iron regulation and has been shown to participate in the assembly of iron-sulfur (Fe-S) clusters under a variety of roles, including modulating persulfide production and directing Fe(II) delivery to the assembly scaffold protein. While the activity and structure of multiple eukaryotic frataxin orthologs have been characterized, the fly ortholog has numerous advantages over other orthologs with regards to protein stability, its activity towards Fe-S cluster assembly and its stability for forming stable proteins partner assemblies. Given the obvious advantages for studying the Drosophila melanogaster frataxin homolog (Dfh) over its orthologs, we have undertaken a structural characterization of apo-Dfh as the first step towards solving the solution structure of the protein alone and in complex with protein partners within the Fe-S cluster assembly pathway.

Transcriptional regulation of the Pseudomonas aeruginosa iron-sulfur cluster assembly pathway by binding of IscR to multiple sites.

Iron-sulfur ([Fe-S]) cluster proteins have essential functions in many biological processes. [Fe-S] homeostasis is crucial for bacterial survival under a wide range of environmental conditions. IscR is a global transcriptional regulator in Pseudomonas aeruginosa; it has been shown to regulate genes involved in [Fe-S] cluster biosynthesis, iron homeostasis, resistance to oxidants, and pathogenicity. Many aspects of the IscR transcriptional regulatory mechanism differ from those of other well-studied systems. This study demonstrates the mechanisms of IscR Type-1 binding to its target sites that mediate the repression of gene expression at the isc operon, nfuA, and tpx. The analysis of IscR binding to multiple binding sites in the promoter region of the isc operon reveals that IscR first binds to the high-affinity site B followed by binding to the low-affinity site A. The results of in vitro IscR binding assays and in vivo analysis of IscR-mediated repression of gene expression support the role of site B as the primary site, while site A has only a minor role in the efficiency of IscR repression of gene expression. Ligation of an [Fe-S] cluster to IscR is required for the binding of IscR to target sites and in vivo repression and stress-induced gene expression. Analysis of Type-1 sites in many bacteria, including P. aeruginosa, indicates that the first and the last three AT-rich bases were among the most highly conserved bases within all analyzed Type-1 sites. Herein, we first propose the putative sequence of P. aeruginosa IscR Type-1 binding motif as 5’AWWSSYRMNNWWWTNNNWSGGNYWW3’. This can benefit further studies in the identification of novel genes under the IscR regulon and the regulatory mechanism model of P. aeruginosa IscR as it contributes to the roles of an [Fe-S] cluster in several biologically important cellular activities.

Mechanistically Driven Control over Cubane Oxo Cluster Catalysts.

Predictive and mechanistically driven access to polynuclear oxo clusters and related materials remains a grand challenge of inorganic chemistry. We here introduce a novel strategy for synthetic control over highly sought-after transition metal {M4O4} cubanes. They attract interest as molecular water oxidation catalysts that combine features of both heterogeneous oxide catalysts and nature's cuboidal {CaMn4O5} center of photosystem II. For the first time, we demonstrate the outstanding structure-directing effect of straightforward inorganic counteranions in solution on the self-assembly of oxo clusters. We introduce a selective counteranion toolbox for the controlled assembly of di(2-pyridyl) ketone (dpk) with M(OAc)2 (M = Co, Ni) precursors into different cubane types. Perchlorate anions provide selective access to type 2 cubanes with the characteristic {H2O-M2(OR)2-OH2} edge-site, such as [Co4(dpy-C{OH}O)4(OAc)2(H2O)2](ClO4)2. Type 1 cubanes with separated polar faces [Co4(dpy-C{OH}O)4(L2)4] n+ (L2 = OAc, Cl, or OAc and H2O) can be tuned with a wide range of other counteranions. The combination of these counteranion sets with Ni(OAc)2 as precursor selectively produces type 2 Co/Ni-mixed or {Ni4O4} cubanes. Systematic mechanistic experiments in combination with computational studies provide strong evidence for type 2 cubane formation through reaction of the key dimeric building block [M2(dpy-C{OH}O)2(H2O)4]2+ with monomers, such as [Co(dpy-C{OH}O)(OAc)(H2O)3]. Furthermore, both experiments and DFT calculations support an energetically favorable type 1 cubane formation pathway via direct head-to-head combination of two [Co2(dpy-C{OH}O)2(OAc)2(H2O)2] dimers. Finally, the visible-light-driven water oxidation activity of type 1 and 2 cubanes with tuned ligand environments was assessed. We pave the way to efficient design concepts in coordination chemistry through ionic control over cluster assembly pathways. Our comprehensive strategy demonstrates how retrosynthetic analyses can be implemented with readily available assembly directing counteranions to provide rapid access to tuned molecular materials.