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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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Further model validation by designing ad hoc Fdx mutants.A, mapping the mutations of Asp11, Glu57, Asp60, and Asp70 on the surface of the Fdx structure. The side chain of Asp74 is also shown. The cluster is represented as red and yellow spheres. B, multiple alignment of Fdx using the same species as selected in Fig. 4 for IscS. The mutated positions are marked by red balloons. Asp70 and Asp74 are completely conserved. C, titrations of 15N-labeled holo-Fdx_D70K (left), holo-Fdx_D11K (middle), and holo-Fdx_E57K/D60K (right) with IscS.
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Figure 6: Further model validation by designing ad hoc Fdx mutants.A, mapping the mutations of Asp11, Glu57, Asp60, and Asp70 on the surface of the Fdx structure. The side chain of Asp74 is also shown. The cluster is represented as red and yellow spheres. B, multiple alignment of Fdx using the same species as selected in Fig. 4 for IscS. The mutated positions are marked by red balloons. Asp70 and Asp74 are completely conserved. C, titrations of 15N-labeled holo-Fdx_D70K (left), holo-Fdx_D11K (middle), and holo-Fdx_E57K/D60K (right) with IscS.

Mentions: Using the predictive power of this holo-Fdx·IscS model, acidic to basic residue mutations were introduced in Fdx at residues Asp70, Asp11, and Glu57/Asp60 (Fig. 6A). Asp11, Glu57/Asp60, and Asp70 are in distinct exposed regions of holo-Fdx but only the latter is involved in interaction in our preliminary holo-Fdx/IscS model. The residues affected have different degrees of conservation (Fig. 6B). Binding between 15N-labeled holo-Fdx_D70K and IscS was not observed even up to 2.5 molar eq of IscS (Fig. 6C). Binding was instead retained as with wild-type holo-Fdx for 15N-labeled holo-Fdx_D11K and holo-Fdx_E57K/D60K. These results allow us to select a unique representative model of the holo-Fdx·IscS complex with excellent statistics (Table 2).


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)

Further model validation by designing ad hoc Fdx mutants.A, mapping the mutations of Asp11, Glu57, Asp60, and Asp70 on the surface of the Fdx structure. The side chain of Asp74 is also shown. The cluster is represented as red and yellow spheres. B, multiple alignment of Fdx using the same species as selected in Fig. 4 for IscS. The mutated positions are marked by red balloons. Asp70 and Asp74 are completely conserved. C, titrations of 15N-labeled holo-Fdx_D70K (left), holo-Fdx_D11K (middle), and holo-Fdx_E57K/D60K (right) with IscS.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 6: Further model validation by designing ad hoc Fdx mutants.A, mapping the mutations of Asp11, Glu57, Asp60, and Asp70 on the surface of the Fdx structure. The side chain of Asp74 is also shown. The cluster is represented as red and yellow spheres. B, multiple alignment of Fdx using the same species as selected in Fig. 4 for IscS. The mutated positions are marked by red balloons. Asp70 and Asp74 are completely conserved. C, titrations of 15N-labeled holo-Fdx_D70K (left), holo-Fdx_D11K (middle), and holo-Fdx_E57K/D60K (right) with IscS.
Mentions: Using the predictive power of this holo-Fdx·IscS model, acidic to basic residue mutations were introduced in Fdx at residues Asp70, Asp11, and Glu57/Asp60 (Fig. 6A). Asp11, Glu57/Asp60, and Asp70 are in distinct exposed regions of holo-Fdx but only the latter is involved in interaction in our preliminary holo-Fdx/IscS model. The residues affected have different degrees of conservation (Fig. 6B). Binding between 15N-labeled holo-Fdx_D70K and IscS was not observed even up to 2.5 molar eq of IscS (Fig. 6C). Binding was instead retained as with wild-type holo-Fdx for 15N-labeled holo-Fdx_D11K and holo-Fdx_E57K/D60K. These results allow us to select a unique representative model of the holo-Fdx·IscS complex with excellent statistics (Table 2).

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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