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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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Quantification of the affinity of IscS·Fdx interaction and effect of Fdx on Fe-S cluster assembly.A, BLI profiles for IscS (at the concentrations indicated) binding to immobilized holo-Fdx. B, BLI profiles showing the displacement of IscS from immobilized holo-Fdx by CyaY (at the concentrations indicated) in the absence of IscU. C, as in B but in the presence of IscU. D, enzymatic Fe-S cluster reconstitution assay on IscU. Cluster assembly was followed by measuring A456 with time. From top to bottom: control with IscS and IscU only (navy), adding 2 μm holo-FDX (green), adding 5 μm CyaY and 10 μm holo-Fdx (orange), adding 5 μm CyaY and 5 μm holo-Fdx (cyan), adding 5 μm CyaY and 2 μm holo-Fdx (blue), and adding 5 μm CyaY (magenta).
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Figure 2: Quantification of the affinity of IscS·Fdx interaction and effect of Fdx on Fe-S cluster assembly.A, BLI profiles for IscS (at the concentrations indicated) binding to immobilized holo-Fdx. B, BLI profiles showing the displacement of IscS from immobilized holo-Fdx by CyaY (at the concentrations indicated) in the absence of IscU. C, as in B but in the presence of IscU. D, enzymatic Fe-S cluster reconstitution assay on IscU. Cluster assembly was followed by measuring A456 with time. From top to bottom: control with IscS and IscU only (navy), adding 2 μm holo-FDX (green), adding 5 μm CyaY and 10 μm holo-Fdx (orange), adding 5 μm CyaY and 5 μm holo-Fdx (cyan), adding 5 μm CyaY and 2 μm holo-Fdx (blue), and adding 5 μm CyaY (magenta).

Mentions: To confirm and quantify this observation, we measured the affinity of holo-Fdx for IscS by BLI. We immobilized holo-Fdx and measured the binding affinity by titration of the protein with increasing concentrations of IscS (Fig. 2A). Holo-Fdx binds to IscS with fast kon and koff rates, which allow evaluation of the Kd at the equilibrium. The Kd value obtained was 1.5 ± 0.4 μm, which is comparable with the Kd between IscU and IscS (1.5 ± 0.3 μm) (19).


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)

Quantification of the affinity of IscS·Fdx interaction and effect of Fdx on Fe-S cluster assembly.A, BLI profiles for IscS (at the concentrations indicated) binding to immobilized holo-Fdx. B, BLI profiles showing the displacement of IscS from immobilized holo-Fdx by CyaY (at the concentrations indicated) in the absence of IscU. C, as in B but in the presence of IscU. D, enzymatic Fe-S cluster reconstitution assay on IscU. Cluster assembly was followed by measuring A456 with time. From top to bottom: control with IscS and IscU only (navy), adding 2 μm holo-FDX (green), adding 5 μm CyaY and 10 μm holo-Fdx (orange), adding 5 μm CyaY and 5 μm holo-Fdx (cyan), adding 5 μm CyaY and 2 μm holo-Fdx (blue), and adding 5 μm CyaY (magenta).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Quantification of the affinity of IscS·Fdx interaction and effect of Fdx on Fe-S cluster assembly.A, BLI profiles for IscS (at the concentrations indicated) binding to immobilized holo-Fdx. B, BLI profiles showing the displacement of IscS from immobilized holo-Fdx by CyaY (at the concentrations indicated) in the absence of IscU. C, as in B but in the presence of IscU. D, enzymatic Fe-S cluster reconstitution assay on IscU. Cluster assembly was followed by measuring A456 with time. From top to bottom: control with IscS and IscU only (navy), adding 2 μm holo-FDX (green), adding 5 μm CyaY and 10 μm holo-Fdx (orange), adding 5 μm CyaY and 5 μm holo-Fdx (cyan), adding 5 μm CyaY and 2 μm holo-Fdx (blue), and adding 5 μm CyaY (magenta).
Mentions: To confirm and quantify this observation, we measured the affinity of holo-Fdx for IscS by BLI. We immobilized holo-Fdx and measured the binding affinity by titration of the protein with increasing concentrations of IscS (Fig. 2A). Holo-Fdx binds to IscS with fast kon and koff rates, which allow evaluation of the Kd at the equilibrium. The Kd value obtained was 1.5 ± 0.4 μm, which is comparable with the Kd between IscU and IscS (1.5 ± 0.3 μm) (19).

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