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Molecular details of the yeast frataxin-Isu1 interaction during mitochondrial Fe-S cluster assembly.

ABSTRACT: Frataxin, a conserved nuclear-encoded mitochondrial protein, plays a direct role in iron-sulfur cluster biosynthesis within the ISC assembly pathway. Humans with frataxin deficiency have Friedreich's ataxia, a neurodegenerative disorder characterized by mitochondrial iron overload and disruption in Fe-S cluster synthesis. Biochemical and genetic studies have shown frataxin interacts with the iron-sulfur cluster assembly scaffold protein (in yeast, there are two, Isu1 and Isu2), indicating frataxin plays a direct role in cluster assembly, possibly by serving as an iron chaperone in the assembly pathway. Here we provide molecular details of how yeast frataxin (Yfh1) interacts with Isu1 as a structural module to improve our understanding of the multiprotein complex assembly that completes Fe-S cluster assembly; this complex also includes the cysteine desulfurase (Nfs1 in yeast) and the accessory protein (Isd11), together in the mitochondria. Thermodynamic binding parameters for protein partner and iron binding were measured for the yeast orthologs using isothermal titration calorimetry. Nuclear magnetic resonance spectroscopy was used to provide the molecular details to understand how Yfh1 interacts with Isu1. X-ray absorption studies were used to electronically and structurally characterize how iron is transferred to Isu1 and then incorporated into an Fe-S cluster. These results were combined with previously published data to generate a structural model for how the Fe-S cluster protein assembly complex can come together to accomplish Fe-S cluster assembly.

SUBMITTER: Cook JD

PROVIDER: S-EPMC3005940 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Molecular details of the yeast frataxin-Isu1 interaction during mitochondrial Fe-S cluster assembly.

Cook Jeremy D JD Kondapalli Kalyan C KC Rawat Swati S Childs William C WC Murugesan Yogapriya Y Dancis Andrew A Stemmler Timothy L TL

Biochemistry 20100914 40

Frataxin, a conserved nuclear-encoded mitochondrial protein, plays a direct role in iron-sulfur cluster biosynthesis within the ISC assembly pathway. Humans with frataxin deficiency have Friedreich's ataxia, a neurodegenerative disorder characterized by mitochondrial iron overload and disruption in Fe-S cluster synthesis. Biochemical and genetic studies have shown frataxin interacts with the iron-sulfur cluster assembly scaffold protein (in yeast, there are two, Isu1 and Isu2), indicating fratax ...[more]

PMID: 20815377

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Project description:Mitochondrial Fe-S cluster biosynthesis is accomplished within yeast utilizing the biophysical attributes of the "Isu1" scaffold assembly protein. As a member of a highly homologous protein family, Isu1 has sequence conservation between orthologs and a conserved ability to assemble [2Fe-2S] clusters. Regardless of species, scaffold orthologs have been shown to exist in both "disordered" and "structured" conformations, a structural architecture that is directly related to conformations utilized during Fe-S cluster assembly. During assembly, the scaffold helps direct the delivery and utilization of Fe(ii) and persulfide substrates to produce [2Fe-2S] clusters, however Zn(ii) binding alters the activity of the scaffold while at the same time stabilizes the protein in its structured state. Additional studies confirm Zn binds to the scaffold's Cys rich active site, and has an impact on the protein's ability to make Fe-S clusters. Understanding the interplay between Fe(ii) and Zn(ii) binding to Isu1 in vitro may help clarify metal loading events that occur during Fe-S cluster assembly in vivo. Here we determine the metal : protein stoichiometry for Isu1 Zn and Fe binding to be 1 : 1 and 2 : 1, respectively. As expected, while Zn binding shifts the Isu1 to its structured state, folding is not influenced by Fe(ii) binding. X-ray absorption spectroscopy (XAS) confirms Zn(ii) binds to the scaffold's cysteine rich active site but Fe(ii) binds at a location distinct from the active site. XAS results show Isu1 binding initially of either Fe(ii) or Zn(ii) does not significantly perturb the metal site structure of alternate metal. XAS confirmed that four scaffold orthologs bind iron as high-spin Fe(ii) at a site composed of ca. 6 oxygen and nitrogen nearest neighbor ligands. Finally, in our report Zn binding dramatically reduces the Fe-S cluster assembly activity of Isu1 even in the presence of frataxin. Given the Fe-binding activity we report for Isu1 and its orthologs here, a possible mechanism involving Fe(ii) transport to the scaffold's active site during cluster assembly has been considered.

Dataset Information

Molecular details of the yeast frataxin-Isu1 interaction during mitochondrial Fe-S cluster assembly.

Publications

Molecular details of the yeast frataxin-Isu1 interaction during mitochondrial Fe-S cluster assembly.

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