Dataset Information

Implications of Membrane Binding by the Fe-S Cluster-Containing N-Terminal Domain in the Drosophila Mitochondrial Replicative DNA Helicase.

ABSTRACT: Recent evidence suggests that iron-sulfur clusters (ISCs) in DNA replicative proteins sense DNA-mediated charge transfer to modulate nuclear DNA replication. In the mitochondrial DNA replisome, only the replicative DNA helicase (mtDNA helicase) from Drosophila melanogaster (Dm) has been shown to contain an ISC in its N-terminal, primase-like domain (NTD). In this report, we confirm the presence of the ISC and demonstrate the importance of a metal cofactor in the structural stability of the Dm mtDNA helicase. Further, we show that the NTD also serves a role in membrane binding. We demonstrate that the NTD binds to asolectin liposomes, which mimic phospholipid membranes, through electrostatic interactions. Notably, membrane binding is more specific with increasing cardiolipin content, which is characteristically high in the mitochondrial inner membrane (MIM). We suggest that the N-terminal domain of the mtDNA helicase interacts with the MIM to recruit mtDNA and initiate mtDNA replication. Furthermore, Dm NUBPL, the known ISC donor for respiratory complex I and a putative donor for Dm mtDNA helicase, was identified as a peripheral membrane protein that is likely to execute membrane-mediated ISC delivery to its target proteins.

SUBMITTER: So M

PROVIDER: S-EPMC8688847 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Publications

Implications of Membrane Binding by the Fe-S Cluster-Containing N-Terminal Domain in the <i>Drosophila</i> Mitochondrial Replicative DNA Helicase.

So Minyoung M Stiban Johnny J Ciesielski Grzegorz L GL Hovde Stacy L SL Kaguni Laurie S LS

Frontiers in genetics 20211207

Recent evidence suggests that iron-sulfur clusters (ISCs) in DNA replicative proteins sense DNA-mediated charge transfer to modulate nuclear DNA replication. In the mitochondrial DNA replisome, only the replicative DNA helicase (mtDNA helicase) from <i>Drosophila melanogaster (Dm)</i> has been shown to contain an ISC in its N-terminal, primase-like domain (NTD). In this report, we confirm the presence of the ISC and demonstrate the importance of a metal cofactor in the structural stability of th ...[more]

PMID: 34950192

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Project description:Frataxin, a mitochondrial protein that is directly involved in regulating cellular iron homeostasis, has been suggested to serve as an iron chaperone during cellular Fe-S cluster biosynthesis. In humans, decreased amounts or impaired function of frataxin causes the autosomal recessive neurodegenerative disorder Friedreich's ataxia. Cellular production of Fe-S clusters is accomplished by the Fe cofactor assembly platform enzymes Isu (eukaryotes) and IscU (prokaryotes). In this report, we have characterized the overall stability and iron binding properties of the Drosophila frataxin homologue (Dfh). Dfh is highly folded with secondary structural elements consistent with the structurally characterized frataxin orthologs. While the melting temperature ( T M approximately 59 degrees C) and chemical stability ([urea] 50% approximately 2.4 M) of Drosophila frataxin, measured using circular dichroism (CD) and fluorescence spectroscopy, closely match values determined for the human ortholog, pure Dfh is more stable against autodegradation than both the human and yeast proteins. The ferrous iron binding affinity ( K d approximately 6.0 microM) and optimal metal to protein stoichiometry (1:1) for Dfh have been measured using isothermal titration calorimetry (ITC). Under anaerobic conditions with salt present, holo-Dfh is a stable iron-loaded protein monomer. Frataxin prevents reactive oxygen species-induced oxidative damage to DNA when presented with both Fe(II) and H 2O 2. Ferrous iron bound to Dfh is high-spin and held in a partially symmetric Fe-(O/N) 6 coordination environment, as determined by X-ray absorption spectroscopy (XAS). Extended X-ray absorption fine structure (EXAFS) simulations indicate the average Fe-O/N bond length in Dfh is 2.13 A, consistent with a ligand geometry constructed by water and carboxylate oxygens most likely supplied in part by surface-exposed conserved acidic residues located on helix 1 and strand 1 in the structurally characterized frataxin orthologs. The iron-dependent binding affinity ( K d approximately 0.21 microM) and optimal holo-Dfh to Isu monomer stoichiometry (1:1) have also been determined using ITC. Finally, frataxin mediates the delivery of Fe(II) to Isu, promoting Fe-S cluster assembly in vitro. The Dfh-assisted assembly of Fe-S clusters occurs with an observed kinetic rate constant ( k obs) of 0.096 min (-1).

Dataset Information

Implications of Membrane Binding by the Fe-S Cluster-Containing N-Terminal Domain in the Drosophila Mitochondrial Replicative DNA Helicase.

Publications

Implications of Membrane Binding by the Fe-S Cluster-Containing N-Terminal Domain in the <i>Drosophila</i> Mitochondrial Replicative DNA Helicase.

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