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

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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.


IscU does not compete with IscS binding. Left: Comparison of the spectra of 15N-labeled IscU (top), the same but after addition of HscB 1:0.4 (middle) and after the further addition of IscS 1:0.4:0.4 (bottom). Right: Comparison of the spectra of 15N-labeled HscB (top), the same but after addition of IscU 1:1 (middle) and after the further addition of IscS 1:1:1 (bottom).
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Figure 6: IscU does not compete with IscS binding. Left: Comparison of the spectra of 15N-labeled IscU (top), the same but after addition of HscB 1:0.4 (middle) and after the further addition of IscS 1:0.4:0.4 (bottom). Right: Comparison of the spectra of 15N-labeled HscB (top), the same but after addition of IscU 1:1 (middle) and after the further addition of IscS 1:1:1 (bottom).

Mentions: Finally, we checked if the HscB/IscS complex is compatible or mutually exclusive with IscU binding. We added unlabeled HscB to 15N labeled IscU (1:0.4 IscU:HscB) and observed the typical chemical shifts expected for binding (Figure 6, Left). We then added unlabeled IscS (1:0.4:0.4) and observed further disappearance of the spectrum. Since this could simply mean a displacement of HscB, we also titrated labeled HscB with unlabeled IscU (1:1), to which we added unlabeled IscS (1:1:1). Also in this case the spectrum disappears almost completely (Figure 6, Right). Titration of HscB with IscS first and then with IscU led to the same result (data not shown). These results conclusively indicate that interaction between HscB and IscU is compatible with IscS binding, a hypothesis not previously considered.


A New Tessera into the Interactome of the isc Operon: A Novel Interaction between HscB and IscS
IscU does not compete with IscS binding. Left: Comparison of the spectra of 15N-labeled IscU (top), the same but after addition of HscB 1:0.4 (middle) and after the further addition of IscS 1:0.4:0.4 (bottom). Right: Comparison of the spectra of 15N-labeled HscB (top), the same but after addition of IscU 1:1 (middle) and after the further addition of IscS 1:1:1 (bottom).
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Related In: Results  -  Collection

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Figure 6: IscU does not compete with IscS binding. Left: Comparison of the spectra of 15N-labeled IscU (top), the same but after addition of HscB 1:0.4 (middle) and after the further addition of IscS 1:0.4:0.4 (bottom). Right: Comparison of the spectra of 15N-labeled HscB (top), the same but after addition of IscU 1:1 (middle) and after the further addition of IscS 1:1:1 (bottom).
Mentions: Finally, we checked if the HscB/IscS complex is compatible or mutually exclusive with IscU binding. We added unlabeled HscB to 15N labeled IscU (1:0.4 IscU:HscB) and observed the typical chemical shifts expected for binding (Figure 6, Left). We then added unlabeled IscS (1:0.4:0.4) and observed further disappearance of the spectrum. Since this could simply mean a displacement of HscB, we also titrated labeled HscB with unlabeled IscU (1:1), to which we added unlabeled IscS (1:1:1). Also in this case the spectrum disappears almost completely (Figure 6, Right). Titration of HscB with IscS first and then with IscU led to the same result (data not shown). These results conclusively indicate that interaction between HscB and IscU is compatible with IscS binding, a hypothesis not previously considered.

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.