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Hybrid Methods in Iron-Sulfur Cluster Biogenesis.

ABSTRACT: Hybrid methods, which combine and integrate several biochemical and biophysical techniques, have rapidly caught up in the last twenty years to provide a way to obtain a fuller description of proteins and molecular complexes with sizes and complexity otherwise not easily affordable. Here, we review the use of a robust hybrid methodology based on a mixture of NMR, SAXS, site directed mutagenesis and molecular docking which we have developed to determine the structure of weakly interacting molecular complexes. We applied this technique to gain insights into the structure of complexes formed amongst proteins involved in the molecular machine, which produces the essential iron-sulfur cluster prosthetic groups. Our results were validated both by X-ray structures and by other groups who adopted the same approach. We discuss the advantages and the limitations of our methodology and propose new avenues, which could improve it.

SUBMITTER: Prischi F

PROVIDER: S-EPMC5346568 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Hybrid Methods in Iron-Sulfur Cluster Biogenesis.

Prischi Filippo F Pastore Annalisa A

Frontiers in molecular biosciences 20170313

Hybrid methods, which combine and integrate several biochemical and biophysical techniques, have rapidly caught up in the last twenty years to provide a way to obtain a fuller description of proteins and molecular complexes with sizes and complexity otherwise not easily affordable. Here, we review the use of a robust hybrid methodology based on a mixture of NMR, SAXS, site directed mutagenesis and molecular docking which we have developed to determine the structure of weakly interacting molecula ...[more]

PMID: 28349052

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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

Hybrid Methods in Iron-Sulfur Cluster Biogenesis.

Publications

Hybrid Methods in Iron-Sulfur Cluster Biogenesis.

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