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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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Model of the holo-Fdx·IscS complex.A, ribbon representations of the holo-Fdx·IscS HADDOCK model at orthogonal orientations. Fdx is shown in brown, the IscS dimer in pale yellow. B, surface electrostatic potentials of holo-Fdx and IscS (red, negative; blue, positive). The interface of the interaction is indicated by the dotted ellipses. C, ribbon representation of the model of the holo-Fdx·IscS·IscU complex IscU is indicated in blue. The model is shown at orthogonal orientations. D, the same as in C but using a surface representation.
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Figure 8: Model of the holo-Fdx·IscS complex.A, ribbon representations of the holo-Fdx·IscS HADDOCK model at orthogonal orientations. Fdx is shown in brown, the IscS dimer in pale yellow. B, surface electrostatic potentials of holo-Fdx and IscS (red, negative; blue, positive). The interface of the interaction is indicated by the dotted ellipses. C, ribbon representation of the model of the holo-Fdx·IscS·IscU complex IscU is indicated in blue. The model is shown at orthogonal orientations. D, the same as in C but using a surface representation.

Mentions: Holo-Fdx binds in a cleft between the two IscS protomers (Fig. 8A) and covers a surface area of 2061.2 ± 57.8 Å2. The interaction is largely electrostatic (Fig. 8B), involving contacts between Asp70, Asp71, and Asp74 of holo-Fdx and Arg220, Arg223, and Arg237 of one IscS protomer. Glu80 and Glu82 of holo-Fdx interact with Arg112 and Arg116 of the second IscS protomer. The binding surface overlaps significantly with CyaY (19) in agreement with the competition experiments. Superimposition of the HADDOCK model of holo-Fdx·IscS with the crystal structure of the IscU·IscS complex (20) shows that IscS can accommodate both holo-Fdx and IscU in a hypothetical ternary holo-Fdx·IscS·IscU complex (Fig. 8C). In such a model, the [2Fe-2S] cluster of holo-Fdx is oriented between the active site cysteine loop of IscS and the cysteine ligands of IscU. The C terminus of holo-Fdx, which contains a histidine and a tyrosine, points toward the interface between IscS and IscU, and could be involved in electron transfer between the [2Fe-2S] cluster of holo-Fdx to the active site cysteine loop of IscS and/or the [2Fe-2S] cluster on IscU. This model provides the first structural insights into the holo-Fdx complex and its importance for cluster formation.


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)

Model of the holo-Fdx·IscS complex.A, ribbon representations of the holo-Fdx·IscS HADDOCK model at orthogonal orientations. Fdx is shown in brown, the IscS dimer in pale yellow. B, surface electrostatic potentials of holo-Fdx and IscS (red, negative; blue, positive). The interface of the interaction is indicated by the dotted ellipses. C, ribbon representation of the model of the holo-Fdx·IscS·IscU complex IscU is indicated in blue. The model is shown at orthogonal orientations. D, the same as in C but using a surface representation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Model of the holo-Fdx·IscS complex.A, ribbon representations of the holo-Fdx·IscS HADDOCK model at orthogonal orientations. Fdx is shown in brown, the IscS dimer in pale yellow. B, surface electrostatic potentials of holo-Fdx and IscS (red, negative; blue, positive). The interface of the interaction is indicated by the dotted ellipses. C, ribbon representation of the model of the holo-Fdx·IscS·IscU complex IscU is indicated in blue. The model is shown at orthogonal orientations. D, the same as in C but using a surface representation.
Mentions: Holo-Fdx binds in a cleft between the two IscS protomers (Fig. 8A) and covers a surface area of 2061.2 ± 57.8 Å2. The interaction is largely electrostatic (Fig. 8B), involving contacts between Asp70, Asp71, and Asp74 of holo-Fdx and Arg220, Arg223, and Arg237 of one IscS protomer. Glu80 and Glu82 of holo-Fdx interact with Arg112 and Arg116 of the second IscS protomer. The binding surface overlaps significantly with CyaY (19) in agreement with the competition experiments. Superimposition of the HADDOCK model of holo-Fdx·IscS with the crystal structure of the IscU·IscS complex (20) shows that IscS can accommodate both holo-Fdx and IscU in a hypothetical ternary holo-Fdx·IscS·IscU complex (Fig. 8C). In such a model, the [2Fe-2S] cluster of holo-Fdx is oriented between the active site cysteine loop of IscS and the cysteine ligands of IscU. The C terminus of holo-Fdx, which contains a histidine and a tyrosine, points toward the interface between IscS and IscU, and could be involved in electron transfer between the [2Fe-2S] cluster of holo-Fdx to the active site cysteine loop of IscS and/or the [2Fe-2S] cluster on IscU. This model provides the first structural insights into the holo-Fdx complex and its importance for cluster formation.

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