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The crystal structure of TrxA(CACA): Insights into the formation of a [2Fe-2S] iron-sulfur cluster in an Escherichia coli thioredoxin mutant.

ABSTRACT: Escherichia coli thioredoxin is a small monomeric protein that reduces disulfide bonds in cytoplasmic proteins. Two cysteine residues present in a conserved CGPC motif are essential for this activity. Recently, we identified mutations of this motif that changed thioredoxin into a homodimer bridged by a [2Fe-2S] iron-sulfur cluster. When exported to the periplasm, these thioredoxin mutants could restore disulfide bond formation in strains lacking the entire periplasmic oxidative pathway. Essential for the assembly of the iron-sulfur was an additional cysteine that replaced the proline at position three of the CGPC motif. We solved the crystalline structure at 2.3 Angstroms for one of these variants, TrxA(CACA). The mutant protein crystallized as a dimer in which the iron-sulfur cluster is replaced by two intermolecular disulfide bonds. The catalytic site, which forms the dimer interface, crystallized in two different conformations. In one of them, the replacement of the CGPC motif by CACA has a dramatic effect on the structure and causes the unraveling of an extended alpha-helix. In both conformations, the second cysteine residue of the CACA motif is surface-exposed, which contrasts with wildtype thioredoxin where the second cysteine of the CXXC motif is buried. This exposure of a pair of vicinal cysteine residues apparently allows thioredoxin to acquire an iron-sulfur cofactor at its active site, and thus a new activity and mechanism of action.

SUBMITTER: Collet JF

PROVIDER: S-EPMC2253358 | biostudies-literature | 2005 Jul

REPOSITORIES: biostudies-literature

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The crystal structure of TrxA(CACA): Insights into the formation of a [2Fe-2S] iron-sulfur cluster in an Escherichia coli thioredoxin mutant.

Collet Jean-Francois JF Peisach Daniel D Bardwell James C A JC Xu Zhaohui Z

Protein science : a publication of the Protein Society 20050701 7

Escherichia coli thioredoxin is a small monomeric protein that reduces disulfide bonds in cytoplasmic proteins. Two cysteine residues present in a conserved CGPC motif are essential for this activity. Recently, we identified mutations of this motif that changed thioredoxin into a homodimer bridged by a [2Fe-2S] iron-sulfur cluster. When exported to the periplasm, these thioredoxin mutants could restore disulfide bond formation in strains lacking the entire periplasmic oxidative pathway. Essentia ...[more]

PMID: 15987909

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Project description:While zinc is an essential trace metal in biology, excess zinc is toxic to organisms. Previous studies have shown that zinc toxicity is associated with disruption of the [4Fe-4S] clusters in various dehydratases in Escherichia coli Here, we report that the intracellular zinc overload in E. coli cells inhibits iron-sulfur cluster biogenesis without affecting the preassembled iron-sulfur clusters in proteins. Among the housekeeping iron-sulfur cluster assembly proteins encoded by the gene cluster iscSUA-hscBA-fdx-iscX in E. coli cells, the scaffold IscU, the iron chaperone IscA, and ferredoxin have strong zinc binding activity in cells, suggesting that intracellular zinc overload inhibits iron-sulfur cluster biogenesis by binding to the iron-sulfur cluster assembly proteins. Mutations of the conserved cysteine residues to serine in IscA, IscU, or ferredoxin completely abolish the zinc binding activity of the proteins, indicating that zinc can compete with iron or iron-sulfur cluster binding in IscA, IscU, and ferredoxin and block iron-sulfur cluster biogenesis. Furthermore, intracellular zinc overload appears to emulate the slow-growth phenotype of the E. coli mutant cells with deletion of the iron-sulfur cluster assembly proteins IscU, IscA, and ferredoxin. Our results suggest that intracellular zinc overload inhibits iron-sulfur cluster biogenesis by targeting the iron-sulfur cluster assembly proteins IscU, IscA, and ferredoxin in E. coli cells.IMPORTANCE Zinc toxicity has been implicated in causing various human diseases. High concentrations of zinc can also inhibit bacterial cell growth. However, the underlying mechanism has not been fully understood. Here, we report that zinc overload in Escherichia coli cells inhibits iron-sulfur cluster biogenesis by targeting specific iron-sulfur cluster assembly proteins. Because iron-sulfur proteins are involved in diverse physiological processes, the zinc-mediated inhibition of iron-sulfur cluster biogenesis could be largely responsible for the zinc-mediated cytotoxicity. Our finding provides new insights on how intracellular zinc overload may inhibit cellular functions in bacteria.

Dataset Information

The crystal structure of TrxA(CACA): Insights into the formation of a [2Fe-2S] iron-sulfur cluster in an Escherichia coli thioredoxin mutant.

Publications

The crystal structure of TrxA(CACA): Insights into the formation of a [2Fe-2S] iron-sulfur cluster in an Escherichia coli thioredoxin mutant.

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