Limits...
A New Tessera into the Interactome of the isc Operon: A Novel Interaction between HscB and IscS

View Article: PubMed Central - PubMed

ABSTRACT

Iron sulfur clusters are essential universal prosthetic groups which can be formed inorganically but, in biology, are bound to proteins and produced enzymatically. Most of the components of the machine that produces the clusters are conserved throughout evolution. In bacteria, they are encoded in the isc operon. Previous reports provide information on the role of specific components but a clear picture of how the whole machine works is still missing. We have carried out a study of the effects of the co-chaperone HscB from the model system E. coli. We document a previously undetected weak interaction between the chaperone HscB and the desulfurase IscS, one of the two main players of the machine. The binding site involves a region of HscB in the longer stem of the approximately L-shaped molecule, whereas the interacting surface of IscS overlaps with the surface previously involved in binding other proteins, such as ferredoxin and frataxin. Our findings provide an entirely new perspective to our comprehension of the role of HscB and propose this protein as a component of the IscS complex.

No MeSH data available.


Competition experiments of HscB with CyaY and Fdx. (Top) Superposition of the HSQC spectra of 100 μM free 15N-labeled HscB (black) and in the complex with unlabelled IscS 1:1 (blue). (Middle) HSQC spectra of 100 μM 15N-labeled HscB in complex with IscS with the addition of CyaY (1:1:3). (Bottom) HSQC spectra of 100 μM 15N-labeled HscB in the presence of IscS and apo-Fdx (1:1:3). The experiments are summarized schematically on the right.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5037179&req=5

Figure 5: Competition experiments of HscB with CyaY and Fdx. (Top) Superposition of the HSQC spectra of 100 μM free 15N-labeled HscB (black) and in the complex with unlabelled IscS 1:1 (blue). (Middle) HSQC spectra of 100 μM 15N-labeled HscB in complex with IscS with the addition of CyaY (1:1:3). (Bottom) HSQC spectra of 100 μM 15N-labeled HscB in the presence of IscS and apo-Fdx (1:1:3). The experiments are summarized schematically on the right.

Mentions: To validate the hypothesis, we titrated a sample containing the HscB/IscS complex with CyaY (up to a 1:1:3 molar ratio) or Fdx (up to a ratio 1:1:3). We observed that introduction of CyaY causes the progressive reappearance of the spectrum of HscB, consistent with almost complete displacement of IscS at 1:1:1 (Figure 5, top and middle panels). Titration of the HscB/IscS complex with Fdx also regenerates the spectrum of HscB but at a higher molar ratio: at a 1:1:3 ratio of IscS/HscB/Fdx, the spectrum of HscB is only partially restored (Figure 5, top and bottom panels). This is in agreement with the relative binding affinities (Prischi et al., 2010b; Yan et al., 2013) and confirms that the interaction between IscS and HscB involves the same site which hosts CyaY and Fdx. The site involves a cleft formed between the two protomers of the IscS dimer and contains PLP and the catalytic center (Cupp-Vickery et al., 2003).


A New Tessera into the Interactome of the isc Operon: A Novel Interaction between HscB and IscS
Competition experiments of HscB with CyaY and Fdx. (Top) Superposition of the HSQC spectra of 100 μM free 15N-labeled HscB (black) and in the complex with unlabelled IscS 1:1 (blue). (Middle) HSQC spectra of 100 μM 15N-labeled HscB in complex with IscS with the addition of CyaY (1:1:3). (Bottom) HSQC spectra of 100 μM 15N-labeled HscB in the presence of IscS and apo-Fdx (1:1:3). The experiments are summarized schematically on the right.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037179&req=5

Figure 5: Competition experiments of HscB with CyaY and Fdx. (Top) Superposition of the HSQC spectra of 100 μM free 15N-labeled HscB (black) and in the complex with unlabelled IscS 1:1 (blue). (Middle) HSQC spectra of 100 μM 15N-labeled HscB in complex with IscS with the addition of CyaY (1:1:3). (Bottom) HSQC spectra of 100 μM 15N-labeled HscB in the presence of IscS and apo-Fdx (1:1:3). The experiments are summarized schematically on the right.
Mentions: To validate the hypothesis, we titrated a sample containing the HscB/IscS complex with CyaY (up to a 1:1:3 molar ratio) or Fdx (up to a ratio 1:1:3). We observed that introduction of CyaY causes the progressive reappearance of the spectrum of HscB, consistent with almost complete displacement of IscS at 1:1:1 (Figure 5, top and middle panels). Titration of the HscB/IscS complex with Fdx also regenerates the spectrum of HscB but at a higher molar ratio: at a 1:1:3 ratio of IscS/HscB/Fdx, the spectrum of HscB is only partially restored (Figure 5, top and bottom panels). This is in agreement with the relative binding affinities (Prischi et al., 2010b; Yan et al., 2013) and confirms that the interaction between IscS and HscB involves the same site which hosts CyaY and Fdx. The site involves a cleft formed between the two protomers of the IscS dimer and contains PLP and the catalytic center (Cupp-Vickery et al., 2003).

View Article: PubMed Central - PubMed

ABSTRACT

Iron sulfur clusters are essential universal prosthetic groups which can be formed inorganically but, in biology, are bound to proteins and produced enzymatically. Most of the components of the machine that produces the clusters are conserved throughout evolution. In bacteria, they are encoded in the isc operon. Previous reports provide information on the role of specific components but a clear picture of how the whole machine works is still missing. We have carried out a study of the effects of the co-chaperone HscB from the model system E. coli. We document a previously undetected weak interaction between the chaperone HscB and the desulfurase IscS, one of the two main players of the machine. The binding site involves a region of HscB in the longer stem of the approximately L-shaped molecule, whereas the interacting surface of IscS overlaps with the surface previously involved in binding other proteins, such as ferredoxin and frataxin. Our findings provide an entirely new perspective to our comprehension of the role of HscB and propose this protein as a component of the IscS complex.

No MeSH data available.