Crystallization and preliminary X-ray crystallographic analysis of<i>Escherichia coli</i>CyaY, a structural homologue of human frataxin

Lee, Myong Gyong; Cho, Seung Je; Yang, Jong Oh; Song, Hyun Kyu; Suh, Se Won

doi:10.1107/s0907444900005916

Cited by 9 publications

(13 citation statements)

References 12 publications

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“…To study the possible interactions of CyaY with proteins of the isc operon, an affinity chromatography approach was chosen. The His 6 -tagged CyaY, expressed and purified as already described (36), was loaded on a gravity flow Ni 2ϩ -charged metal chelating column, and in a first experiment, an E. coli cell-free extract (ϳ400 mg) prepared from a 3-liter culture of BL21(DE3)-RIL cells overexpressing the whole isc operon was passed over the CyaY-loaded column. Because all of the cellular proteins were not His-tagged, proteins retained on the column and eluted together with CyaY are those that interact with CyaY specifically.…”

Section: Resultsmentioning

confidence: 99%

“…Expression and Purification of Proteins-Recombinant E. coli CyaY containing a C-terminal His 6 tag was expressed and purified as previously described with some minor modifications (36). Following metal-chelate chromatography on nickel-nitrilotriacetic acid resin (Amersham Biosciences) a gel filtration on a HiLoad 16/60 Superdex 75 prep-grade column (Amersham Biosciences) was performed (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 3 mM DTT).…”

Section: Methodsmentioning

confidence: 99%

“…Recombinant E. coli IscU (non-His-tagged), E. coli His-tagged IscU, E. coli His-tagged IscS (both N-terminal His-tagged), E. coli IscS (nonHis-tagged), and E. coli IscS C328S (non-His-tagged) were expressed and purified as previously described (38 -40, 42, 43 Preparation of Cell-free Extracts-E. coli BL21(DE3)-RIL harboring plasmid pRKISC, E. coli BL21(DE3) harboring plasmid pTrcIscU and E. coli BL21(DE3) harboring plasmid pET-22b-CyaY were grown as previously described (36,38,44). Overexpression of proteins of the isc operon, of non-His-tagged IscU and of His-tagged CyaY was induced by addition of 0.5 mM isopropyl ␤-D-thiogalactopyranoside.…”

Section: Methodsmentioning

confidence: 99%

“…Interestingly, during the preparation of this manuscript, it was reported that a double knock-out in cyaY and yggX in Salmonella enterica resulted in defects in [Fe-S] cluster metabolism (32). Structural analysis of E. coli CyaY (x-ray structure at 1.4 Å resolution and NMR structure) and yeast and human frataxin revealed a common protein fold for all frataxin homologs (33)(34)(35)(36). NMR spectroscopic studies on CyaY showed that it is able to bind iron in both Fe 2ϩ and Fe 3ϩ oxidation states through conserved Asp and Glu residues in the same region of the protein as in the case of human frataxin (34 …”

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confidence: 98%

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Iron-Sulfur Cluster Biosynthesis

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Choudens

Sanakis

et al. 2006

Journal of Biological Chemistry

160

113

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The biogenesis of iron-sulfur [Fe-S] clusters requires the coordinated delivery of both iron and sulfide. Sulfide is provided by cysteine desulfurases that use L-cysteine as sulfur source. So far, the physiological iron donor has not been clearly identified. CyaY, the bacterial ortholog of frataxin, an iron binding protein thought to be involved in iron-sulfur cluster formation in eukaryotes, is a good candidate because it was shown to bind iron. Iron-sulfur [Fe-S] clusters are ubiquitous and evolutionary ancient prosthetic groups that are required to sustain fundamental life processes. They are involved in electron transfer, substrate binding/activation, iron/sulfur storage, regulation of gene expression, and enzyme activity reactions (1). Formation of intracellular [Fe-S] clusters does not occur spontaneously but requires a complex biosynthetic machinery. In Escherichia coli three different types of [Fe-S] cluster biosynthesis systems have been identified so far, namely the iron-sulfur cluster, sulfur mobilization, and cysteine sulfinate desulfinase systems (2, 3). These different machineries have in common the involvement of a cysteine desulfurase that allows utilization of cysteine as source of sulfur atoms Important questions related to [Fe-S] cluster biosynthesis include: (i) the molecular mechanism by which iron and sulfide are assembled on the scaffold protein; (ii) how accessory proteins (chaperones in particular) participate in the process; and (iii) how the cluster is transferred from the scaffold to an apo target protein. Another essential question is the identity of iron and sulfur donors for the formation of [Fe-S] clusters. Whereas L-cysteine has been identified as the ultimate source of sulfur, the question "Where does the iron come from?" still remains unanswered. Whereas some preliminary answers have been provided to most of the above issues, very little is known regarding the last question. It is simply assumed that, because of its toxicity, iron has to be stored and transported by proteins from which it can be mobilized for assembly of iron sites. In bacteria, IscA and YggX, which were shown to be able to bind iron and to be, to some extent, involved in [Fe-S] metabolism, are potential candidates requiring further investigations (9 -12). However, this is controversial because IscA was proposed to be an [Fe-S] scaffold protein (5, 7), whereas a recent report could not establish iron binding to YggX (13).More information concerning a putative iron donor protein is available in eukaryotic systems. In eukaryotes, [Fe-S] cluster assembly requires two biosynthetic protein machineries. One is localized in the mitochondria and functions in the assembly of all cellular [Fe-S] proteins, whereas the other one is cytosolic, specifically involved in the maturation of cytosolic and nuclear [Fe-S] (15); (ii) the yeast frataxin (Yfh1p) is involved in the regulation of iron homeostasis (16,17) and is required for maturation of [Fe-S] cluster containing proteins (18), and its inactivation results in i...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 98%

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Iron-Sulfur Cluster Biosynthesis

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Choudens

Sanakis

et al. 2006

Journal of Biological Chemistry

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113

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“…Overexpression, purification, and crystallization of the E. coli CyaY protein with a six-histidine tag at the C terminus were reported elsewhere (14). The dynamic light scattering analysis was performed with a Model DynaPro-801 instrument from Protein Solutions (Charlottesville, VA) as described (15).…”

Section: Methodsmentioning

confidence: 99%

Crystal structure of Escherichia coli CyaY protein reveals a previously unidentified fold for the evolutionarily conserved frataxin family

Cho

Lee

Yang

et al. 2000

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

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Friedreich ataxia is an autosomal recessive neurodegenerative disease caused by defects in the FRDA gene, which encodes a mitochondrial protein called frataxin. Frataxin is evolutionarily conserved, with homologs identified in mammals, worms, yeast, and bacteria. The CyaY proteins of ␥-purple bacteria are believed to be closely related to the ancestor of frataxin. In this study, we have determined the crystal structure of the CyaY protein from Escherichia coli at 1.4-Å resolution. It reveals a protein fold consisting of a six-stranded antiparallel ␤-sheet flanked on one side by two ␣-helices. This fold is likely to be shared by all members of the conserved frataxin family. This study also provides a framework for the interpretation of disease-associated mutations in frataxin and for understanding the possible functions of this protein family.

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Mapping iron binding sites on human frataxin: implications for cluster assembly on the ISU Fe–S cluster scaffold protein

2008

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Frataxin is an iron binding mitochondrial matrix protein that has been shown to mediate iron delivery during iron-sulfur cluster and heme biosynthesis. There is a high degree of structural homology for frataxin proteins from diverse sources, and all possess an anionic surface defined by acidic residues. In the human protein these residues principally lie on a surface defined by the alpha1 helix and beta1 sheet and the impact of multiple substitutions of these carboxylate residues on iron binding is described. Full-length human frataxin has previously been shown to undergo self-cleavage to produce a truncated form both in vitro and in vivo. This truncated protein has been shown to bind approximately seven iron centers that are presumably associated with the acidic patch. Relative to this native protein, the stoichiometry decreases according to the number and sites of mutations. Nevertheless, the iron-dependent binding affinity of each frataxin derivative to the iron-sulfur cluster scaffold protein ISU is found to be similar to that of native frataxin, as defined by isothermal titration calorimetry experiments, requiring only one iron center to promote nanomolar binding. While frataxins from various cell types appear to bind differing numbers of iron centers, the physiologically relevant number of bound irons appears to be small, with significantly higher binding affinity following complex formation with partner proteins (micromolar compared with nanomolar binding). By contrast, in reconstitution assays for frataxin-promoted [2Fe-2S](2+) cluster assembly on ISU, one derivative does display a modestly lower reconstitution rate. The overall consensus from these data is to consider a pool of potential sites that can stably bind an iron center when bridged to a variety of physiological targets.

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Crystallization and preliminary X-ray crystallographic analysis ofEscherichia coliCyaY, a structural homologue of human frataxin

Cited by 9 publications

References 12 publications

Iron-Sulfur Cluster Biosynthesis

Iron-Sulfur Cluster Biosynthesis

Crystal structure of Escherichia coli CyaY protein reveals a previously unidentified fold for the evolutionarily conserved frataxin family

Mapping iron binding sites on human frataxin: implications for cluster assembly on the ISU Fe–S cluster scaffold protein

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