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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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SAXS analysis.A, experimental SAXS data of the IscS and holo-Fdx·IscS binary complex are displayed as dots with error bars (gray), whereas curves computed from ab initio and crystallographic models (PDB 1P3W for IscS and HADDOCK model for holo-Fdx·IscS) are given as solid and dashed lines, respectively. The fit from OLIGOMER for the Iscs·Fdx binary complex with a χ value of 1.04 (yielding 80% of the complex and 20% of free components) is shown as dash-dot lines. The logarithm of the scattering intensity (I) is plotted as a function of the momentum transfer “s”. The successive curves are displaced down appropriately for better visualization. Distance distribution functions are shown in the insert. B, ab initio bead models of IscS (top) and holo-Fdx·IscS (bottom) (gray semitransparent spheres) superimposed with the crystal structure of IscS dimer and the NMR model of holo-Fdx·IscS binary complex. Crystallographic models of IscS and Fdx molecules are displayed as yellow and red Cα traces, respectively.
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Figure 7: SAXS analysis.A, experimental SAXS data of the IscS and holo-Fdx·IscS binary complex are displayed as dots with error bars (gray), whereas curves computed from ab initio and crystallographic models (PDB 1P3W for IscS and HADDOCK model for holo-Fdx·IscS) are given as solid and dashed lines, respectively. The fit from OLIGOMER for the Iscs·Fdx binary complex with a χ value of 1.04 (yielding 80% of the complex and 20% of free components) is shown as dash-dot lines. The logarithm of the scattering intensity (I) is plotted as a function of the momentum transfer “s”. The successive curves are displaced down appropriately for better visualization. Distance distribution functions are shown in the insert. B, ab initio bead models of IscS (top) and holo-Fdx·IscS (bottom) (gray semitransparent spheres) superimposed with the crystal structure of IscS dimer and the NMR model of holo-Fdx·IscS binary complex. Crystallographic models of IscS and Fdx molecules are displayed as yellow and red Cα traces, respectively.

Mentions: We acquired SAXS data for IscS alone and for the holo-Fdx·IscS complex in solution (Table 3 and Fig. 7A). The estimated apparent molecular mass (MMexp) and hydrated particle volume (Vp) for IscS agrees with the presence of a dimer in accordance with previous SAXS results (19). The MMexp and excluded volume of the holo-Fdx·IscS complex are clearly different from those of the isolated IscS and correspond to a stoichiometry of 2:2 for the binary complex. The overall parameters (Rg = 31.0 Å, Dmax = 112 Å) of holo-Fdx·IscS complex are close to those of the CyaY·IscS complex (Rg = 31.1 Å, Dmax = 109 Å) and differ significantly from those of the IscU·IscS complex (Rg = 35.0 Å, Dmax = 121 Å) excluding the possibility that Fdx binds on the periphery of the IscS dimer.


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)

SAXS analysis.A, experimental SAXS data of the IscS and holo-Fdx·IscS binary complex are displayed as dots with error bars (gray), whereas curves computed from ab initio and crystallographic models (PDB 1P3W for IscS and HADDOCK model for holo-Fdx·IscS) are given as solid and dashed lines, respectively. The fit from OLIGOMER for the Iscs·Fdx binary complex with a χ value of 1.04 (yielding 80% of the complex and 20% of free components) is shown as dash-dot lines. The logarithm of the scattering intensity (I) is plotted as a function of the momentum transfer “s”. The successive curves are displaced down appropriately for better visualization. Distance distribution functions are shown in the insert. B, ab initio bead models of IscS (top) and holo-Fdx·IscS (bottom) (gray semitransparent spheres) superimposed with the crystal structure of IscS dimer and the NMR model of holo-Fdx·IscS binary complex. Crystallographic models of IscS and Fdx molecules are displayed as yellow and red Cα traces, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: SAXS analysis.A, experimental SAXS data of the IscS and holo-Fdx·IscS binary complex are displayed as dots with error bars (gray), whereas curves computed from ab initio and crystallographic models (PDB 1P3W for IscS and HADDOCK model for holo-Fdx·IscS) are given as solid and dashed lines, respectively. The fit from OLIGOMER for the Iscs·Fdx binary complex with a χ value of 1.04 (yielding 80% of the complex and 20% of free components) is shown as dash-dot lines. The logarithm of the scattering intensity (I) is plotted as a function of the momentum transfer “s”. The successive curves are displaced down appropriately for better visualization. Distance distribution functions are shown in the insert. B, ab initio bead models of IscS (top) and holo-Fdx·IscS (bottom) (gray semitransparent spheres) superimposed with the crystal structure of IscS dimer and the NMR model of holo-Fdx·IscS binary complex. Crystallographic models of IscS and Fdx molecules are displayed as yellow and red Cα traces, respectively.
Mentions: We acquired SAXS data for IscS alone and for the holo-Fdx·IscS complex in solution (Table 3 and Fig. 7A). The estimated apparent molecular mass (MMexp) and hydrated particle volume (Vp) for IscS agrees with the presence of a dimer in accordance with previous SAXS results (19). The MMexp and excluded volume of the holo-Fdx·IscS complex are clearly different from those of the isolated IscS and correspond to a stoichiometry of 2:2 for the binary complex. The overall parameters (Rg = 31.0 Å, Dmax = 112 Å) of holo-Fdx·IscS complex are close to those of the CyaY·IscS complex (Rg = 31.1 Å, Dmax = 109 Å) and differ significantly from those of the IscU·IscS complex (Rg = 35.0 Å, Dmax = 121 Å) excluding the possibility that Fdx binds on the periphery of the IscS dimer.

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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