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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.

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Identification of the component(s) responsible for slowing down the reaction. (A) Effect of the individual components. Cluster formation rates on Fdx in the presence of IscS and IscU (control, orange), HscA (red), HscB (green), or HscA/HscB (cyan). The experiment is repeated in the absence (left) and presence of ATP (right). (B) Time course of cluster formation on Fdx in the presence of IscS, IscU, DTT with Cys and Fe2+ in the presence of increasing concentrations of HscB. The curves correspond to no HscB (red), 3 μM (blue), 5 μM (green), 8 μM (cyan), 10 μM (purple), 20 μM (gray), and 40 μM (orange). Right: Corresponding rates. (C) Cluster formation rates on IscU in the presence of IscS, DTT with Cys, and Fe2+. The curves correspond to no HscB (red), HscB 5 μM (blue), 10 μM (green), 20 μM (purple), 25 μM (cyan), 30 μM (gray), and 40 μM (orange). Right: Corresponding rates. (D) Cluster formation rates on aconitase in the presence of IscS, IscU, DTT with Cys, Fe2+. The curves correspond to no HscB (red) and HscB 1 μM (blue), 5 μM (green), 10 μM (purple), 15 μM (cyan), 20 μM (gray), and 40 μM (orange). Right: Corresponding rates. (E) Quantification of alanine formation. Alanine production by IscS (left) and in the presence of HscB (right).
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Figure 2: Identification of the component(s) responsible for slowing down the reaction. (A) Effect of the individual components. Cluster formation rates on Fdx in the presence of IscS and IscU (control, orange), HscA (red), HscB (green), or HscA/HscB (cyan). The experiment is repeated in the absence (left) and presence of ATP (right). (B) Time course of cluster formation on Fdx in the presence of IscS, IscU, DTT with Cys and Fe2+ in the presence of increasing concentrations of HscB. The curves correspond to no HscB (red), 3 μM (blue), 5 μM (green), 8 μM (cyan), 10 μM (purple), 20 μM (gray), and 40 μM (orange). Right: Corresponding rates. (C) Cluster formation rates on IscU in the presence of IscS, DTT with Cys, and Fe2+. The curves correspond to no HscB (red), HscB 5 μM (blue), 10 μM (green), 20 μM (purple), 25 μM (cyan), 30 μM (gray), and 40 μM (orange). Right: Corresponding rates. (D) Cluster formation rates on aconitase in the presence of IscS, IscU, DTT with Cys, Fe2+. The curves correspond to no HscB (red) and HscB 1 μM (blue), 5 μM (green), 10 μM (purple), 15 μM (cyan), 20 μM (gray), and 40 μM (orange). Right: Corresponding rates. (E) Quantification of alanine formation. Alanine production by IscS (left) and in the presence of HscB (right).

Mentions: To understand the factors which determine inhibition of HscB and HscA, we explored the effects of each component by introducing them individually and in pair in the absence and in the presence of ATP (Figure 2A). We used again Fdx as the reporter, 1 μM of IscS and 8 μM of IscU, in the presence of 250 μM Cys, 25 μM Fe2+, and 10 mM Mg2+. In the absence of ATP, introduction of HscA (10 μM) does not produce appreciable effects on the enzymatic rates. Addition of HscB (10 μM) remarkably inhibits the rates, while co-addition of HscA and HscB produces a further drop. In the presence of ATP, addition of HscA increases the rates in a comparable way than when adding only ATP. Addition of HscB, alone or together with HscA and ATP appreciably reduces the rates. This tells us that the effect is mainly due to HscB, whether in the presence or absence of HscA.


A New Tessera into the Interactome of the isc Operon: A Novel Interaction between HscB and IscS
Identification of the component(s) responsible for slowing down the reaction. (A) Effect of the individual components. Cluster formation rates on Fdx in the presence of IscS and IscU (control, orange), HscA (red), HscB (green), or HscA/HscB (cyan). The experiment is repeated in the absence (left) and presence of ATP (right). (B) Time course of cluster formation on Fdx in the presence of IscS, IscU, DTT with Cys and Fe2+ in the presence of increasing concentrations of HscB. The curves correspond to no HscB (red), 3 μM (blue), 5 μM (green), 8 μM (cyan), 10 μM (purple), 20 μM (gray), and 40 μM (orange). Right: Corresponding rates. (C) Cluster formation rates on IscU in the presence of IscS, DTT with Cys, and Fe2+. The curves correspond to no HscB (red), HscB 5 μM (blue), 10 μM (green), 20 μM (purple), 25 μM (cyan), 30 μM (gray), and 40 μM (orange). Right: Corresponding rates. (D) Cluster formation rates on aconitase in the presence of IscS, IscU, DTT with Cys, Fe2+. The curves correspond to no HscB (red) and HscB 1 μM (blue), 5 μM (green), 10 μM (purple), 15 μM (cyan), 20 μM (gray), and 40 μM (orange). Right: Corresponding rates. (E) Quantification of alanine formation. Alanine production by IscS (left) and in the presence of HscB (right).
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Figure 2: Identification of the component(s) responsible for slowing down the reaction. (A) Effect of the individual components. Cluster formation rates on Fdx in the presence of IscS and IscU (control, orange), HscA (red), HscB (green), or HscA/HscB (cyan). The experiment is repeated in the absence (left) and presence of ATP (right). (B) Time course of cluster formation on Fdx in the presence of IscS, IscU, DTT with Cys and Fe2+ in the presence of increasing concentrations of HscB. The curves correspond to no HscB (red), 3 μM (blue), 5 μM (green), 8 μM (cyan), 10 μM (purple), 20 μM (gray), and 40 μM (orange). Right: Corresponding rates. (C) Cluster formation rates on IscU in the presence of IscS, DTT with Cys, and Fe2+. The curves correspond to no HscB (red), HscB 5 μM (blue), 10 μM (green), 20 μM (purple), 25 μM (cyan), 30 μM (gray), and 40 μM (orange). Right: Corresponding rates. (D) Cluster formation rates on aconitase in the presence of IscS, IscU, DTT with Cys, Fe2+. The curves correspond to no HscB (red) and HscB 1 μM (blue), 5 μM (green), 10 μM (purple), 15 μM (cyan), 20 μM (gray), and 40 μM (orange). Right: Corresponding rates. (E) Quantification of alanine formation. Alanine production by IscS (left) and in the presence of HscB (right).
Mentions: To understand the factors which determine inhibition of HscB and HscA, we explored the effects of each component by introducing them individually and in pair in the absence and in the presence of ATP (Figure 2A). We used again Fdx as the reporter, 1 μM of IscS and 8 μM of IscU, in the presence of 250 μM Cys, 25 μM Fe2+, and 10 mM Mg2+. In the absence of ATP, introduction of HscA (10 μM) does not produce appreciable effects on the enzymatic rates. Addition of HscB (10 μM) remarkably inhibits the rates, while co-addition of HscA and HscB produces a further drop. In the presence of ATP, addition of HscA increases the rates in a comparable way than when adding only ATP. Addition of HscB, alone or together with HscA and ATP appreciably reduces the rates. This tells us that the effect is mainly due to HscB, whether in the presence or absence of HscA.

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.


Related in: MedlinePlus