Human anamorsin binds [2Fe–2S] clusters with unique electronic properties

Banci, Lucia; Ciofi‐Baffoni, Simone; Mikołajczyk, Maciej; Winkelmann, Julia; Bill, Eckhard; Pandelia, Maria-Eirini

doi:10.1007/s00775-013-1033-1

Cited by 52 publications

(67 citation statements)

References 50 publications

(73 reference statements)

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“…Ciapin1 is an early acting factor in the maturation of newly synthesized [4Fe-4S] clusters in the CIA pathway (43,44). Ciapin1 itself contains two bound Fe-S clusters that are thought to function in the transfer of reducing equivalents from the flavoprotein Ndor1 to downstream components of the CIA (45,46). We confirmed the presence of the Glrx3⅐Ciapin1 complex in cells by detecting endogenous Ciapin1 in Glrx3-FLAG immunoprecipitates (Fig.…”

Section: Glrx3 Forms Homodimers Independent Of Bound Fe-s Clusters-prsupporting

confidence: 56%

A Glutaredoxin·BolA Complex Serves as an Iron-Sulfur Cluster Chaperone for the Cytosolic Cluster Assembly Machinery

Frey

Palenchar

Wildemann

et al. 2016

Journal of Biological Chemistry

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Cells contain hundreds of proteins that require iron cofactors for activity. Iron cofactors are synthesized in the cell, but the pathways involved in distributing heme, iron-sulfur clusters, and ferrous/ferric ions to apoproteins remain incompletely defined. In particular, cytosolic monothiol glutaredoxins and BolA-like proteins have been identified as [2Fe-2S]-coordinating complexes in vitro and iron-regulatory proteins in fungi, but it is not clear how these proteins function in mammalian systems or how this complex might affect Fe-S proteins or the cytosolic Fe-S assembly machinery. To explore these questions, we use quantitative immunoprecipitation and live cell proximitydependent biotinylation to monitor interactions between Glrx3, BolA2, and components of the cytosolic iron-sulfur cluster assembly system. We characterize cytosolic Glrx3⅐BolA2 as a Although hundreds of cellular proteins require iron-containing cofactors for activity (1), the machinery responsible for distributing these cofactors remains relatively obscure. Separate systems must exist for both the mitochondrial and the cytosolic/nuclear compartments and systems must selectively distribute ferrous iron ions, iron-sulfur (Fe-S) centers, and heme.Recent studies indicate that the poly(rC)-binding proteins are involved in the distribution of ferrous iron to ferritin, the principal iron storage protein, and to mono-and dinuclear iron enzymes in the cytosol of mammalian cells (2-4). Studies in both bakers' yeast and vertebrates indicate that many proteins are involved in the assembly and distribution of Fe-S clusters in the cytosol (5-7). These proteins are structurally and functionally conserved across many species. One protein class, the monothiol glutaredoxins, has been functionally implicated in the trafficking of both ionic iron and Fe-S clusters (8 -12).Monothiol glutaredoxins are members of the thioredoxin (Trx) 3 -fold family of proteins. Most members of the Trx family utilize a dithiol active site to catalyze the oxido-reduction of thiol-disulfide residues. In contrast, monothiol glutaredoxins contain a Cys-Gly-Phe-Ser active site that lacks catalytic activity and instead coordinates a [2Fe-2S] cluster via the active site cysteine and the sulfhydryl residue of a molecule of glutathione, which is non-covalently bound adjacent to the glutaredoxin active site (10,(13)(14)(15). In vitro analysis of this Fe-S-containing species indicates that two glutathione-bound glutaredoxin proteins can coordinate a single [2Fe-2S] cluster as a bridging complex. In eukaryotes, distinct monothiol glutaredoxins are expressed in the mitochondria and cytosol. Genetic evidence suggests that mitochondrial glutaredoxins are involved in the transfer of newly assembled Fe-S clusters to recipient apoproteins (8,9,16,17). Cytosolic monothiol glutaredoxins differ from their mitochondrial paralogs in that they contain an amino-terminal Trx-like domain followed by one or more glutaredoxin domains. Studies in fungi suggest these proteins are involved in iron homeostasis.Th...

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Section: Glrx3 Forms Homodimers Independent Of Bound Fe-s Clusters-prsupporting

confidence: 56%

A Glutaredoxin·BolA Complex Serves as an Iron-Sulfur Cluster Chaperone for the Cytosolic Cluster Assembly Machinery

Frey

Palenchar

Wildemann

et al. 2016

Journal of Biological Chemistry

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“…We previously reported that the WT Dre2 protein contains one [2Fe2S] cluster and one [4Fe4S] cluster by EPR analysis (11). Although this finding has been supported by more recent studies (1214), some other studies on anamorsin, the hDre2 homolog, claimed that these two cysteine motifs independently bind a [2Fe2S] cluster in a mutually exclusive manner (19). To resolve the discrepancy, we decided to perform site-directed mutagenesis by mutating each of the cysteine residues to alanine, specifically aiming to disrupt one cluster while not affecting the other.…”

Section: Resultsmentioning

confidence: 61%

EPR studies of wild type and mutant Dre2 identify essential [2Fe–-2S] and [4Fe–-4S] clusters and their cysteine ligands

et al. 2016

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Yeast Dre2 (anamorsin or CIAPIN1) is an essential component for cytosolic Fe/S cluster biosynthesis. The C-terminal domain contains eight evolutionarily conserved cysteine residues, and we previously demonstrated that the yeast Dre2 overexpressed in Escherichia coli contains one binuclear ([2Fe2S]) cluster and one tetranuclear ([4Fe4S]) cluster. In this study, we replaced each conserved cysteine with alanine and analyzed the effects by Electron Paramagnetic Resonance. Although the C311A mutant lacked both signals, our data clearly suggest that the [2Fe2S] cluster is ligated to Cys252, Cys263, Cys266 and Cys268, whereas the [4Fe4S] cluster is ligated to Cys311, Cys314, Cys322 and Cys325. By simulation analysis of the C263A and C322A data, we obtained the g-values for the [4Fe4S] cluster (g x,y,z = 1.830, 1.947 and 2.018) and for the [2Fe2S] cluster (g x,y,z =1.919, 1.962 and 2.001). We also observed spinspin interaction between the two clusters, suggesting their close proximity. Chemically reconstituted Dre2 showed air sensitivity of the [4Fe4S] cluster converting to a [2Fe2S] cluster. Furthermore, using a yeast shuffle strain, we demonstrated for the first time that each of the Cys FeS cluster ligands with the exception of C252 is essential, indicating that both Dre2 clusters are needed for cell viability.

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“…The physiological relevance of the interface of hGRX5 interacting with hISCA1 and hISCA2 was verified by investigating the interaction of hGRX5 with the nonphysiological partner anamorsin, which is a human [2Fe-2S] protein involved in the cytosolic iron-sulfur protein assembly machinery (25,31,32). Specifically, when titrating 15 N-labeled apo hGRX5 with unlabeled [2Fe-2S]-anamorsin no significant chemical shift changes are observed (Fig.…”

Section: It Results That (I) the [2fe-2s]mentioning

confidence: 99%

[2Fe-2S] cluster transfer in iron–sulfur protein biogenesis

Banci

Brancaccio

Ciofi‐Baffoni

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Monothiol glutaredoxins play a crucial role in iron-sulfur (Fe/S) protein biogenesis. Essentially all of them can coordinate a [2Fe-2S] cluster and have been proposed to mediate the transfer of clusters from scaffold proteins to target apo proteins, possibly by acting as cluster transfer proteins. The molecular basis of cluster transfer from monothiol glutaredoxins to target proteins is a fundamental, but still unresolved, aspect to be defined in Fe/S protein biogenesis. In mitochondria monothiol glutaredoxin 5 (GRX5) is involved in the maturation of all cellular Fe/S proteins and participates in cellular iron regulation. Here we show that the structural plasticity of the dimeric state of the [2Fe-2S] bound form of human GRX5 (holo hGRX5) is the crucial factor that allows an efficient cluster transfer to the partner proteins human ISCA1 and ISCA2 by a specific protein-protein recognition mechanism. Holo hGRX5 works as a metallochaperone preventing the [2Fe-2S] cluster to be released in solution in the presence of physiological concentrations of glutathione and forming a transient, cluster-mediated protein-protein intermediate with two physiological protein partners receiving the [2Fe-2S] cluster. The cluster transfer mechanism defined here may extend to other mitochondrial [2Fe-2S] target proteins.Fe/S protein maturation | [2Fe-2S] cluster transfer mechanism | monothiol Grxs | NMR G lutaredoxins (Grxs) and glutathione (GSH) are universally distributed among all organisms, and they have been shown to play a fundamental role in iron-sulfur (Fe/S) protein biogenesis (1-5). Specifically, the [2Fe-2S]-bound forms of monothiol Grxs and a [2Fe-2S]-glutathione complex are the species suggested to be responsible for trafficking [2Fe-2S] clusters within the cell (6-9). The current working model is that in the cell monothiol Grxs receive a [2Fe-2S] cluster from the scaffold protein ISCU (where de novo synthesis of the [2Fe-2S] cluster occurs) and transfer it to specific targeting proteins, which then facilitate Fe/S cluster insertion into the final acceptor apo protein (7, 10, 11). Another possible cluster transfer mechanism, which has been proposed (8), hypothesizes the cellular presence of a [2Fe-2S](GS) 4 complex, which could transiently store [2Fe-2S] clusters, facilitate cluster exchange with the cellular Fe/S cluster biosynthesis machineries, and regulate the biosynthesis of Fe/S clusters. However, a drawback of the latter model is that all of the Fe/S cellular trafficking processes will result to be protein-independent and therefore highly unspecific, thus potentially inflicting severe cellular damage.The mitochondrial, monothiol glutaredoxin 5 protein (GRX5) belongs to the core part of the mitochondrial Fe/S cluster (ISC) assembly system (10, 12, 13), is required in the maturation of all cellular [2Fe-2S] and [4Fe-4S] proteins (11), and participates in cellular iron regulation (14). Human GRX5 in vitro binds a [2Fe-2S] cluster (15) and yeast GRX5, which in vivo and in vitro binds a [2Fe-2S] cluster (11), has been...

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Human anamorsin binds [2Fe–2S] clusters with unique electronic properties

Cited by 52 publications

References 50 publications

A Glutaredoxin·BolA Complex Serves as an Iron-Sulfur Cluster Chaperone for the Cytosolic Cluster Assembly Machinery

A Glutaredoxin·BolA Complex Serves as an Iron-Sulfur Cluster Chaperone for the Cytosolic Cluster Assembly Machinery

EPR studies of wild type and mutant Dre2 identify essential [2Fe–-2S] and [4Fe–-4S] clusters and their cysteine ligands

[2Fe-2S] cluster transfer in iron–sulfur protein biogenesis

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