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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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IscS mutants designed to test interaction with Fdx and their positions in an IscS alignment.Top, IscS backbone structure with the side chains of residues mutated indicated explicitly: R39E/W45E (orange), K101E/K105E (red), R112E/R116E (blue), R220E/R223E/R225E (green), I314E/M315E (magenta), E334S/R340E (cyan), D346K/E347K (black). Bottom, sequence alignment of representative species from bacteria to primates. The signal peptides are omitted in the eukaryotic sequences that start with the homology to the prokaryotic orthologues. The mutated positions are indicated with balloons in the same color coding as used in the structure.
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Figure 4: IscS mutants designed to test interaction with Fdx and their positions in an IscS alignment.Top, IscS backbone structure with the side chains of residues mutated indicated explicitly: R39E/W45E (orange), K101E/K105E (red), R112E/R116E (blue), R220E/R223E/R225E (green), I314E/M315E (magenta), E334S/R340E (cyan), D346K/E347K (black). Bottom, sequence alignment of representative species from bacteria to primates. The signal peptides are omitted in the eukaryotic sequences that start with the homology to the prokaryotic orthologues. The mutated positions are indicated with balloons in the same color coding as used in the structure.

Mentions: To further characterize the surface of IscS interacting with holo-Fdx we tested the ability of ad hoc designed IscS mutants to bind 15N-labeled holo-Fdx by NMR. We initially tested IscS_R39E/W45E, IscS_K101E/K105E, IscS_R220E/R223E/R225E, IscS_D346K/E347K, IscS_I314E/M315E, and IscS_E334S/R340S (Fig. 4). Of these mutants, only IscS_R220E/R223E/R225E did not bind to holo-Fdx: when titrated with IscS_R220E/R223E/R225E, no change was observed in the spectrum of holo-Fdx up to 4 molar eq as expected if mutation abolishes binding (Table 1). A similar behavior was observed for CyaY in the complex with IscS in agreement with CyaY and Fdx sharing the same binding site (19). Consistently, holo-Fdx, like CyaY, is an acidic protein (pI 4.49). The residues found to interact with IscS are all part of an acidic patch on 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)

IscS mutants designed to test interaction with Fdx and their positions in an IscS alignment.Top, IscS backbone structure with the side chains of residues mutated indicated explicitly: R39E/W45E (orange), K101E/K105E (red), R112E/R116E (blue), R220E/R223E/R225E (green), I314E/M315E (magenta), E334S/R340E (cyan), D346K/E347K (black). Bottom, sequence alignment of representative species from bacteria to primates. The signal peptides are omitted in the eukaryotic sequences that start with the homology to the prokaryotic orthologues. The mutated positions are indicated with balloons in the same color coding as used in the structure.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: IscS mutants designed to test interaction with Fdx and their positions in an IscS alignment.Top, IscS backbone structure with the side chains of residues mutated indicated explicitly: R39E/W45E (orange), K101E/K105E (red), R112E/R116E (blue), R220E/R223E/R225E (green), I314E/M315E (magenta), E334S/R340E (cyan), D346K/E347K (black). Bottom, sequence alignment of representative species from bacteria to primates. The signal peptides are omitted in the eukaryotic sequences that start with the homology to the prokaryotic orthologues. The mutated positions are indicated with balloons in the same color coding as used in the structure.
Mentions: To further characterize the surface of IscS interacting with holo-Fdx we tested the ability of ad hoc designed IscS mutants to bind 15N-labeled holo-Fdx by NMR. We initially tested IscS_R39E/W45E, IscS_K101E/K105E, IscS_R220E/R223E/R225E, IscS_D346K/E347K, IscS_I314E/M315E, and IscS_E334S/R340S (Fig. 4). Of these mutants, only IscS_R220E/R223E/R225E did not bind to holo-Fdx: when titrated with IscS_R220E/R223E/R225E, no change was observed in the spectrum of holo-Fdx up to 4 molar eq as expected if mutation abolishes binding (Table 1). A similar behavior was observed for CyaY in the complex with IscS in agreement with CyaY and Fdx sharing the same binding site (19). Consistently, holo-Fdx, like CyaY, is an acidic protein (pI 4.49). The residues found to interact with IscS are all part of an acidic patch on 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