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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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Details on the six clusters obtained by HADDOCK calculations. The corresponding energies are shown in Table 2.
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Figure 5: Details on the six clusters obtained by HADDOCK calculations. The corresponding energies are shown in Table 2.

Mentions: To validate or reject this solution, we designed and tested an IscS_R112E/R116E. The chemical shift changes on Fdx are in the fast-exchange regime as opposed to slow-exchange for wild-type IscS. The peaks are still observable up to 0.6 molecular eq indicating that holo-Fdx binds IscS_R112E/R116E but with reduced affinity. We used this result to refine the input AIRs for the HADDOCK run obtaining six clusters (Fig. 5). Clusters 3 and 6 did not satisfy the AIRs for IscS Arg112 and Arg116 and so could be rejected. Clusters 1, 4, and 5 varied considerably with the position and rotational orientation of Fdx with respect to IscS. Clusters 4 and 5 were significantly less energetically favorable than Cluster 1 in terms of restraint violations (Table 2). Cluster 2 gave similar statistics as Cluster 1. The position and orientation of Fdx between Cluster 1 and Cluster 2 were similar, with root mean square deviation between the two lowest energy representatives of these clusters of 0.45 Å. Cluster 1 was taken as representative of the complex as this solution has better statistics.


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)

Details on the six clusters obtained by HADDOCK calculations. The corresponding energies are shown in Table 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Details on the six clusters obtained by HADDOCK calculations. The corresponding energies are shown in Table 2.
Mentions: To validate or reject this solution, we designed and tested an IscS_R112E/R116E. The chemical shift changes on Fdx are in the fast-exchange regime as opposed to slow-exchange for wild-type IscS. The peaks are still observable up to 0.6 molecular eq indicating that holo-Fdx binds IscS_R112E/R116E but with reduced affinity. We used this result to refine the input AIRs for the HADDOCK run obtaining six clusters (Fig. 5). Clusters 3 and 6 did not satisfy the AIRs for IscS Arg112 and Arg116 and so could be rejected. Clusters 1, 4, and 5 varied considerably with the position and rotational orientation of Fdx with respect to IscS. Clusters 4 and 5 were significantly less energetically favorable than Cluster 1 in terms of restraint violations (Table 2). Cluster 2 gave similar statistics as Cluster 1. The position and orientation of Fdx between Cluster 1 and Cluster 2 were similar, with root mean square deviation between the two lowest energy representatives of these clusters of 0.45 Å. Cluster 1 was taken as representative of the complex as this solution has better statistics.

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