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Ferredoxin competes with bacterial frataxin in binding to the desulfurase IscS.

Yan R, Konarev PV, Iannuzzi C, Adinolfi S, Roche B, Kelly G, Simon L, Martin SR, Py B, Barras F, Svergun DI, Pastore A - J. Biol. Chem. (2013)

Bottom Line: Here, we have characterized the interaction using a combination of biophysical tools and mutagenesis.By modeling the Fdx·IscS complex based on experimental restraints we show that Fdx competes for the binding site of CyaY, the bacterial ortholog of frataxin and sits in a cavity close to the enzyme active site.Our data provide the first structural insights into the role of Fdx in cluster assembly.

View Article: PubMed Central - PubMed

Affiliation: MRC National Institute for Medical Research, The Ridgeway, London NW7 1AA, United Kingdom.

ABSTRACT
The bacterial iron-sulfur cluster (isc) operon is an essential machine that is highly conserved from bacteria to primates and responsible for iron-sulfur cluster biogenesis. Among its components are the genes for the desulfurase IscS that provides sulfur for cluster formation, and a specialized ferredoxin (Fdx) whose role is still unknown. Preliminary evidence suggests that IscS and Fdx interact but nothing is known about the binding site and the role of the interaction. Here, we have characterized the interaction using a combination of biophysical tools and mutagenesis. By modeling the Fdx·IscS complex based on experimental restraints we show that Fdx competes for the binding site of CyaY, the bacterial ortholog of frataxin and sits in a cavity close to the enzyme active site. By in vivo mutagenesis in bacteria we prove the importance of the surface of interaction for cluster formation. Our data provide the first structural insights into the role of Fdx in cluster assembly.

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In vivo effect of mutated versions of the fdx gene in IscR maturation. β-Galactosidase activity from wild-type (WT) and Δfdx strains (Δfdx) carrying the PiscR::lacZ fusion and transformed with pBAD, pFdxWT, pFdxD70K, or pFdxD70KD74K was measured in cells grown in LB supplemented with 50 μg/ml of ampicillin and 0.2% arabinose. Results shown are the mean of triplicate experiments.
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Figure 9: In vivo effect of mutated versions of the fdx gene in IscR maturation. β-Galactosidase activity from wild-type (WT) and Δfdx strains (Δfdx) carrying the PiscR::lacZ fusion and transformed with pBAD, pFdxWT, pFdxD70K, or pFdxD70KD74K was measured in cells grown in LB supplemented with 50 μg/ml of ampicillin and 0.2% arabinose. Results shown are the mean of triplicate experiments.

Mentions: Finally, we used an in vivo assay to test the importance of the residues involved in Fdx·IscS interaction in Fe-S biogenesis. We employed the E. coli Fe-S cluster-dependent transcriptional regulator IscR as reporter for Fe-S protein maturation and used an E. coli strain carrying the lacZ reporter gene fused to a gene whose expression is repressed by the Fe-S bound form of IscR, iscR (PiscR::lacZ) (31). As previously reported, introduction of fdx deletion leads to a defect in PiscR repression (Fig. 9) (37). This defect was recovered by complementing the Δfdx strain with a wild-type pFdxWT plasmid and the pFdxD70K but not with the pFdxD70KD74K mutant (Fig. 9). These results indicate that mutation of these residues causes a defect in Fdx activity in Fe-S cluster biogenesis and highlight their importance in vivo.


Ferredoxin competes with bacterial frataxin in binding to the desulfurase IscS.

Yan R, Konarev PV, Iannuzzi C, Adinolfi S, Roche B, Kelly G, Simon L, Martin SR, Py B, Barras F, Svergun DI, Pastore A - J. Biol. Chem. (2013)

In vivo effect of mutated versions of the fdx gene in IscR maturation. β-Galactosidase activity from wild-type (WT) and Δfdx strains (Δfdx) carrying the PiscR::lacZ fusion and transformed with pBAD, pFdxWT, pFdxD70K, or pFdxD70KD74K was measured in cells grown in LB supplemented with 50 μg/ml of ampicillin and 0.2% arabinose. Results shown are the mean of triplicate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750173&req=5

Figure 9: In vivo effect of mutated versions of the fdx gene in IscR maturation. β-Galactosidase activity from wild-type (WT) and Δfdx strains (Δfdx) carrying the PiscR::lacZ fusion and transformed with pBAD, pFdxWT, pFdxD70K, or pFdxD70KD74K was measured in cells grown in LB supplemented with 50 μg/ml of ampicillin and 0.2% arabinose. Results shown are the mean of triplicate experiments.
Mentions: Finally, we used an in vivo assay to test the importance of the residues involved in Fdx·IscS interaction in Fe-S biogenesis. We employed the E. coli Fe-S cluster-dependent transcriptional regulator IscR as reporter for Fe-S protein maturation and used an E. coli strain carrying the lacZ reporter gene fused to a gene whose expression is repressed by the Fe-S bound form of IscR, iscR (PiscR::lacZ) (31). As previously reported, introduction of fdx deletion leads to a defect in PiscR repression (Fig. 9) (37). This defect was recovered by complementing the Δfdx strain with a wild-type pFdxWT plasmid and the pFdxD70K but not with the pFdxD70KD74K mutant (Fig. 9). These results indicate that mutation of these residues causes a defect in Fdx activity in Fe-S cluster biogenesis and highlight their importance in vivo.

Bottom Line: Here, we have characterized the interaction using a combination of biophysical tools and mutagenesis.By modeling the Fdx·IscS complex based on experimental restraints we show that Fdx competes for the binding site of CyaY, the bacterial ortholog of frataxin and sits in a cavity close to the enzyme active site.Our data provide the first structural insights into the role of Fdx in cluster assembly.

View Article: PubMed Central - PubMed

Affiliation: MRC National Institute for Medical Research, The Ridgeway, London NW7 1AA, United Kingdom.

ABSTRACT
The bacterial iron-sulfur cluster (isc) operon is an essential machine that is highly conserved from bacteria to primates and responsible for iron-sulfur cluster biogenesis. Among its components are the genes for the desulfurase IscS that provides sulfur for cluster formation, and a specialized ferredoxin (Fdx) whose role is still unknown. Preliminary evidence suggests that IscS and Fdx interact but nothing is known about the binding site and the role of the interaction. Here, we have characterized the interaction using a combination of biophysical tools and mutagenesis. By modeling the Fdx·IscS complex based on experimental restraints we show that Fdx competes for the binding site of CyaY, the bacterial ortholog of frataxin and sits in a cavity close to the enzyme active site. By in vivo mutagenesis in bacteria we prove the importance of the surface of interaction for cluster formation. Our data provide the first structural insights into the role of Fdx in cluster assembly.

Show MeSH
Related in: MedlinePlus