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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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Chemical shift mapping of the holo-Fdx surface interacting with IscS.A, representative examples of the chemical shift perturbation observed in 1H,15N-SOFAST HMQC spectra of 15N/2H-labeled holo-Fdx upon titration with IscS to 0 (black), 0.1 (red), 0.2 (green), 0.3 (blue), and 0.4 equivalents (magenta). B, the Δ chemical shift at 0.3 equivalents of IscS per holo-Fdx residue. Residues denoted by a star are broadened at 0.3 equivalents. The indole Nϵ1Hϵ1 of Trp77 is denoted with an chevron. C, mapping the interaction on the holo-Fdx surface. The side chains exhibiting the largest Δ chemical shifts as denoted by the shaded rectangle in B are explicitly shown in green.
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Figure 3: Chemical shift mapping of the holo-Fdx surface interacting with IscS.A, representative examples of the chemical shift perturbation observed in 1H,15N-SOFAST HMQC spectra of 15N/2H-labeled holo-Fdx upon titration with IscS to 0 (black), 0.1 (red), 0.2 (green), 0.3 (blue), and 0.4 equivalents (magenta). B, the Δ chemical shift at 0.3 equivalents of IscS per holo-Fdx residue. Residues denoted by a star are broadened at 0.3 equivalents. The indole Nϵ1Hϵ1 of Trp77 is denoted with an chevron. C, mapping the interaction on the holo-Fdx surface. The side chains exhibiting the largest Δ chemical shifts as denoted by the shaded rectangle in B are explicitly shown in green.

Mentions: We mapped the surface of holo-Fdx interacting with IscS by NMR exploiting the chemical shift perturbation observed in the spectrum of holo-Fdx upon titration with IscS. Holo-Fdx was deuterated to attenuate the contribution of dipole-dipole interactions on the transverse relaxation. Chemical shift perturbations were readily observed up to 0.4 eq of IscS for Ile54, Val55, Gln68, Glu69, Asp70, Asp71, Met72, Leu73, Asp74, Lys75, Ala76, Trp77, Gly78, Leu79, Glu80, Glu82 and for the Nϵ1Hϵ1 indole group of Trp77 (Fig. 3A). Met72, Leu73, and Glu80 were completely broadened by 0.3 eq. At 0.5 eq and above the signals of holo-Fdx broadened. The largest variations of chemical shifts were observed for Gln68–Glu82 (Fig. 3B), suggesting that this region of holo-Fdx interacts with IscS directly. These residues are in helix 2 and the following loop of holo-Fdx (Fig. 3C) and form an exposed acidic patch on the surface of holo-Fdx.


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)

Chemical shift mapping of the holo-Fdx surface interacting with IscS.A, representative examples of the chemical shift perturbation observed in 1H,15N-SOFAST HMQC spectra of 15N/2H-labeled holo-Fdx upon titration with IscS to 0 (black), 0.1 (red), 0.2 (green), 0.3 (blue), and 0.4 equivalents (magenta). B, the Δ chemical shift at 0.3 equivalents of IscS per holo-Fdx residue. Residues denoted by a star are broadened at 0.3 equivalents. The indole Nϵ1Hϵ1 of Trp77 is denoted with an chevron. C, mapping the interaction on the holo-Fdx surface. The side chains exhibiting the largest Δ chemical shifts as denoted by the shaded rectangle in B are explicitly shown in green.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Chemical shift mapping of the holo-Fdx surface interacting with IscS.A, representative examples of the chemical shift perturbation observed in 1H,15N-SOFAST HMQC spectra of 15N/2H-labeled holo-Fdx upon titration with IscS to 0 (black), 0.1 (red), 0.2 (green), 0.3 (blue), and 0.4 equivalents (magenta). B, the Δ chemical shift at 0.3 equivalents of IscS per holo-Fdx residue. Residues denoted by a star are broadened at 0.3 equivalents. The indole Nϵ1Hϵ1 of Trp77 is denoted with an chevron. C, mapping the interaction on the holo-Fdx surface. The side chains exhibiting the largest Δ chemical shifts as denoted by the shaded rectangle in B are explicitly shown in green.
Mentions: We mapped the surface of holo-Fdx interacting with IscS by NMR exploiting the chemical shift perturbation observed in the spectrum of holo-Fdx upon titration with IscS. Holo-Fdx was deuterated to attenuate the contribution of dipole-dipole interactions on the transverse relaxation. Chemical shift perturbations were readily observed up to 0.4 eq of IscS for Ile54, Val55, Gln68, Glu69, Asp70, Asp71, Met72, Leu73, Asp74, Lys75, Ala76, Trp77, Gly78, Leu79, Glu80, Glu82 and for the Nϵ1Hϵ1 indole group of Trp77 (Fig. 3A). Met72, Leu73, and Glu80 were completely broadened by 0.3 eq. At 0.5 eq and above the signals of holo-Fdx broadened. The largest variations of chemical shifts were observed for Gln68–Glu82 (Fig. 3B), suggesting that this region of holo-Fdx interacts with IscS directly. These residues are in helix 2 and the following loop of holo-Fdx (Fig. 3C) and form an exposed acidic patch on the surface of holo-Fdx.

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