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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.

No MeSH data available.


Related in: MedlinePlus

Optimizing the concentrations of the Isc components on the formation of the cluster on Fdx. (A) Left: Scan of the IscU concentration. Rates of cluster formation on 50 μM Fdx in the presence of 1 μM IscS, 1 μM HscA, 1 μM HscB, 3 mM DTT, 250 μM cys, 150 μM ATP, 25 μM Fe2+, and 10 μM Mg2+. IscU was progressively 1 μM (green), 5 μM (violet), 10 μM (cyan), and 20 μM (orange). Right: Corresponding rates estimated from the slope of the initial part of the curves after the lag time. (B) Left: Scan of the HscB concentration. The concentrations of IscU and HscA are fixed at 8 and 2 μM. HscB is varied from 0 μM (blue), 5 μM (violet), 10 μM (cyan), 15 μM (orange), 20 μM (pale blue), and 30 μM (red). The other components are as in the previous experiment. Right: Corresponding initial rates. (C) Left: Scan of the HscA concentration. The concentrations of IscU and HscB are fixed to 8 and 1 μM. The other components are as above. HscA was varied from 0 μM (blue) to 1 μM (pale blue), 5 μM (green), 10 μM (violet), 15 μM (orange), 20 μM (cyan), and 30 μM (red). Right: Corresponding initial rates. (D) Left: Scan of the ATP concentration in the absence of Mg2+. IscU, HscA, and HscB are fixed at 8, 2, and 3 μM, respectively. ATP is varied from 0 μM (green) to 50 μM (cyan), 150 μM (violet), 500 μM (red), and 1 mM (pale blue). Right: The same as the left panel but with the addition of 10 mM Mg2+.
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Figure 1: Optimizing the concentrations of the Isc components on the formation of the cluster on Fdx. (A) Left: Scan of the IscU concentration. Rates of cluster formation on 50 μM Fdx in the presence of 1 μM IscS, 1 μM HscA, 1 μM HscB, 3 mM DTT, 250 μM cys, 150 μM ATP, 25 μM Fe2+, and 10 μM Mg2+. IscU was progressively 1 μM (green), 5 μM (violet), 10 μM (cyan), and 20 μM (orange). Right: Corresponding rates estimated from the slope of the initial part of the curves after the lag time. (B) Left: Scan of the HscB concentration. The concentrations of IscU and HscA are fixed at 8 and 2 μM. HscB is varied from 0 μM (blue), 5 μM (violet), 10 μM (cyan), 15 μM (orange), 20 μM (pale blue), and 30 μM (red). The other components are as in the previous experiment. Right: Corresponding initial rates. (C) Left: Scan of the HscA concentration. The concentrations of IscU and HscB are fixed to 8 and 1 μM. The other components are as above. HscA was varied from 0 μM (blue) to 1 μM (pale blue), 5 μM (green), 10 μM (violet), 15 μM (orange), 20 μM (cyan), and 30 μM (red). Right: Corresponding initial rates. (D) Left: Scan of the ATP concentration in the absence of Mg2+. IscU, HscA, and HscB are fixed at 8, 2, and 3 μM, respectively. ATP is varied from 0 μM (green) to 50 μM (cyan), 150 μM (violet), 500 μM (red), and 1 mM (pale blue). Right: The same as the left panel but with the addition of 10 mM Mg2+.

Mentions: Increasing IscU concentrations (1, 5, 10, 20 μM) enhanced the reconstitution rate and shortened the lag phase, reaching a plateau above 5–10 μM (Figure 1A). This is reasonable since increase of IscU concentration facilitates formation of the IscS-IscU complex and accelerates the reaction. We thus adopted 8 μM concentrations of IscU in the following experiments to ensure high activity but, at the same time, avoid contributions from holo-IscU to absorbance. Progressive increase of HscB concentration caused a clear reduction of the reaction rates (Figure 1B). A 3 μM HscB concentration was adopted in the following experiments to ensure a small excess of protein with respect to IscS in a range of concentrations where inhibition is not dominant. The ATPase activity of HscA was independently checked by a spectrophotometric method that allows the quantification of inorganic phosphate release (data not shown). Variation of the HscA concentration in the range 1–5 μM led to no change of the cluster formation rates. Above ~5 μM, we observed a deep decrease of the rates (Figure 1C). A 2 μM concentration was thus adopted in the following measurement to ensure a small excess of protein with respect to IscS. Finally, we screened the effect of ATP varying it from 0 to 1 mM in the absence and in the presence of 10 mM Mg2+. When no Mg2+ was added we observed a dramatic reduction of the rates up to almost complete inhibition of the reaction (Figure 1D). The effect was drastically reduced and practically abolished at 10 mM Mg2+ which are the concentrations typically used for this assay because close to the cellular conditions.


A New Tessera into the Interactome of the isc Operon: A Novel Interaction between HscB and IscS
Optimizing the concentrations of the Isc components on the formation of the cluster on Fdx. (A) Left: Scan of the IscU concentration. Rates of cluster formation on 50 μM Fdx in the presence of 1 μM IscS, 1 μM HscA, 1 μM HscB, 3 mM DTT, 250 μM cys, 150 μM ATP, 25 μM Fe2+, and 10 μM Mg2+. IscU was progressively 1 μM (green), 5 μM (violet), 10 μM (cyan), and 20 μM (orange). Right: Corresponding rates estimated from the slope of the initial part of the curves after the lag time. (B) Left: Scan of the HscB concentration. The concentrations of IscU and HscA are fixed at 8 and 2 μM. HscB is varied from 0 μM (blue), 5 μM (violet), 10 μM (cyan), 15 μM (orange), 20 μM (pale blue), and 30 μM (red). The other components are as in the previous experiment. Right: Corresponding initial rates. (C) Left: Scan of the HscA concentration. The concentrations of IscU and HscB are fixed to 8 and 1 μM. The other components are as above. HscA was varied from 0 μM (blue) to 1 μM (pale blue), 5 μM (green), 10 μM (violet), 15 μM (orange), 20 μM (cyan), and 30 μM (red). Right: Corresponding initial rates. (D) Left: Scan of the ATP concentration in the absence of Mg2+. IscU, HscA, and HscB are fixed at 8, 2, and 3 μM, respectively. ATP is varied from 0 μM (green) to 50 μM (cyan), 150 μM (violet), 500 μM (red), and 1 mM (pale blue). Right: The same as the left panel but with the addition of 10 mM Mg2+.
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Figure 1: Optimizing the concentrations of the Isc components on the formation of the cluster on Fdx. (A) Left: Scan of the IscU concentration. Rates of cluster formation on 50 μM Fdx in the presence of 1 μM IscS, 1 μM HscA, 1 μM HscB, 3 mM DTT, 250 μM cys, 150 μM ATP, 25 μM Fe2+, and 10 μM Mg2+. IscU was progressively 1 μM (green), 5 μM (violet), 10 μM (cyan), and 20 μM (orange). Right: Corresponding rates estimated from the slope of the initial part of the curves after the lag time. (B) Left: Scan of the HscB concentration. The concentrations of IscU and HscA are fixed at 8 and 2 μM. HscB is varied from 0 μM (blue), 5 μM (violet), 10 μM (cyan), 15 μM (orange), 20 μM (pale blue), and 30 μM (red). The other components are as in the previous experiment. Right: Corresponding initial rates. (C) Left: Scan of the HscA concentration. The concentrations of IscU and HscB are fixed to 8 and 1 μM. The other components are as above. HscA was varied from 0 μM (blue) to 1 μM (pale blue), 5 μM (green), 10 μM (violet), 15 μM (orange), 20 μM (cyan), and 30 μM (red). Right: Corresponding initial rates. (D) Left: Scan of the ATP concentration in the absence of Mg2+. IscU, HscA, and HscB are fixed at 8, 2, and 3 μM, respectively. ATP is varied from 0 μM (green) to 50 μM (cyan), 150 μM (violet), 500 μM (red), and 1 mM (pale blue). Right: The same as the left panel but with the addition of 10 mM Mg2+.
Mentions: Increasing IscU concentrations (1, 5, 10, 20 μM) enhanced the reconstitution rate and shortened the lag phase, reaching a plateau above 5–10 μM (Figure 1A). This is reasonable since increase of IscU concentration facilitates formation of the IscS-IscU complex and accelerates the reaction. We thus adopted 8 μM concentrations of IscU in the following experiments to ensure high activity but, at the same time, avoid contributions from holo-IscU to absorbance. Progressive increase of HscB concentration caused a clear reduction of the reaction rates (Figure 1B). A 3 μM HscB concentration was adopted in the following experiments to ensure a small excess of protein with respect to IscS in a range of concentrations where inhibition is not dominant. The ATPase activity of HscA was independently checked by a spectrophotometric method that allows the quantification of inorganic phosphate release (data not shown). Variation of the HscA concentration in the range 1–5 μM led to no change of the cluster formation rates. Above ~5 μM, we observed a deep decrease of the rates (Figure 1C). A 2 μM concentration was thus adopted in the following measurement to ensure a small excess of protein with respect to IscS. Finally, we screened the effect of ATP varying it from 0 to 1 mM in the absence and in the presence of 10 mM Mg2+. When no Mg2+ was added we observed a dramatic reduction of the rates up to almost complete inhibition of the reaction (Figure 1D). The effect was drastically reduced and practically abolished at 10 mM Mg2+ which are the concentrations typically used for this assay because close to the cellular conditions.

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