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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.


Probing a direct interaction between IscS and HscB. (A) MST data for the IscS and HscB interaction. Data are plotted as normalized signal changes as a function of the HscB concentration. The average of three experiments is reported. (B) SDS-PAGE of IscS, HscB, and IscS:HscB mixture in the presence of a cross-linker (BS3). (C) HSQC-NMR spectra of 15N-labeled HscB 100 μM (black) and 15N-labeled HscB 100 μM in the presence of IscS 1:1 (red). (D) Mapping the interaction surface on HscB (PDB code: 1FPO). Left: the residues showing chemical shift perturbation at a 1:1 molar ratio are shown in green, those which broaden or disappear in magenta. For comparison, the residues identified as involved in IscU binding are shown in blue. Right: residues giving cross-saturation with IscS at the same molar ratio (gray).
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Figure 3: Probing a direct interaction between IscS and HscB. (A) MST data for the IscS and HscB interaction. Data are plotted as normalized signal changes as a function of the HscB concentration. The average of three experiments is reported. (B) SDS-PAGE of IscS, HscB, and IscS:HscB mixture in the presence of a cross-linker (BS3). (C) HSQC-NMR spectra of 15N-labeled HscB 100 μM (black) and 15N-labeled HscB 100 μM in the presence of IscS 1:1 (red). (D) Mapping the interaction surface on HscB (PDB code: 1FPO). Left: the residues showing chemical shift perturbation at a 1:1 molar ratio are shown in green, those which broaden or disappear in magenta. For comparison, the residues identified as involved in IscU binding are shown in blue. Right: residues giving cross-saturation with IscS at the same molar ratio (gray).

Mentions: Although not previously reported, a possible working hypothesis to explain the observed effect is that HscB binds also to IscS and that this interferes with the desulfuration step. To test the hypothesis, we carried out pull-down and size exclusion chromatography assays but both proved inconclusive, as it can be expected for weak complexes. We thus used microscale thermophoresis (MST) which is a sensitive means to measure weak interactions (Jerabek-Willemsen et al., 2014). The thermophoretic movement of the fluorescently labeled IscS was assessed by measuring the fluorescence distribution inside a capillary and the microscopic temperature gradient generated by a laser. The binding curve was obtained by plotting the fluorescence vs. the logarithm of the progressively diluted concentrations of HscB (Figure 3A). The data obtained by different values of light emitting diode (LED) resulted in a dissociation constant (KD) of 10–30 μM assuming a 1:1 stoichiometry. These affinities are low but comparable to those observed for other components of the isc machine (Prischi et al., 2010b) and, more importantly, to that observed for the IscU/HscB interaction (~10 μM, Hoff et al., 2000). To confirm the interaction with an independent method, we used chemical cross-linking, a technique able to capture weak or transient protein-protein interactions (Watson et al., 2012). We added bis[sulfosuccinimidyl]suberate, a cross-linking agent that reacts with primary amino groups, to a mixture containing HscB and IscS. SDS-PAGE revealed the presence of a new species at around 65 KDa that corresponds to HscB-IscS covalently bound (Figure 3B).


A New Tessera into the Interactome of the isc Operon: A Novel Interaction between HscB and IscS
Probing a direct interaction between IscS and HscB. (A) MST data for the IscS and HscB interaction. Data are plotted as normalized signal changes as a function of the HscB concentration. The average of three experiments is reported. (B) SDS-PAGE of IscS, HscB, and IscS:HscB mixture in the presence of a cross-linker (BS3). (C) HSQC-NMR spectra of 15N-labeled HscB 100 μM (black) and 15N-labeled HscB 100 μM in the presence of IscS 1:1 (red). (D) Mapping the interaction surface on HscB (PDB code: 1FPO). Left: the residues showing chemical shift perturbation at a 1:1 molar ratio are shown in green, those which broaden or disappear in magenta. For comparison, the residues identified as involved in IscU binding are shown in blue. Right: residues giving cross-saturation with IscS at the same molar ratio (gray).
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Figure 3: Probing a direct interaction between IscS and HscB. (A) MST data for the IscS and HscB interaction. Data are plotted as normalized signal changes as a function of the HscB concentration. The average of three experiments is reported. (B) SDS-PAGE of IscS, HscB, and IscS:HscB mixture in the presence of a cross-linker (BS3). (C) HSQC-NMR spectra of 15N-labeled HscB 100 μM (black) and 15N-labeled HscB 100 μM in the presence of IscS 1:1 (red). (D) Mapping the interaction surface on HscB (PDB code: 1FPO). Left: the residues showing chemical shift perturbation at a 1:1 molar ratio are shown in green, those which broaden or disappear in magenta. For comparison, the residues identified as involved in IscU binding are shown in blue. Right: residues giving cross-saturation with IscS at the same molar ratio (gray).
Mentions: Although not previously reported, a possible working hypothesis to explain the observed effect is that HscB binds also to IscS and that this interferes with the desulfuration step. To test the hypothesis, we carried out pull-down and size exclusion chromatography assays but both proved inconclusive, as it can be expected for weak complexes. We thus used microscale thermophoresis (MST) which is a sensitive means to measure weak interactions (Jerabek-Willemsen et al., 2014). The thermophoretic movement of the fluorescently labeled IscS was assessed by measuring the fluorescence distribution inside a capillary and the microscopic temperature gradient generated by a laser. The binding curve was obtained by plotting the fluorescence vs. the logarithm of the progressively diluted concentrations of HscB (Figure 3A). The data obtained by different values of light emitting diode (LED) resulted in a dissociation constant (KD) of 10–30 μM assuming a 1:1 stoichiometry. These affinities are low but comparable to those observed for other components of the isc machine (Prischi et al., 2010b) and, more importantly, to that observed for the IscU/HscB interaction (~10 μM, Hoff et al., 2000). To confirm the interaction with an independent method, we used chemical cross-linking, a technique able to capture weak or transient protein-protein interactions (Watson et al., 2012). We added bis[sulfosuccinimidyl]suberate, a cross-linking agent that reacts with primary amino groups, to a mixture containing HscB and IscS. SDS-PAGE revealed the presence of a new species at around 65 KDa that corresponds to HscB-IscS covalently bound (Figure 3B).

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.