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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.


Identification of the surface of interaction on IscS by mutagenesis. Position of the mutations on the structure of IscS (PDB code: 3LVL) and HSQC-NMR spectra of 15N-labeled HscB 100 μM (black) and in the presence of the IscS mutants (1:1). Spectra in red are not affected, spectra in orange show the disappearance of few peaks because of the formation of a complex IscS:HscB. The same color coding is used in the structure panel.
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Figure 4: Identification of the surface of interaction on IscS by mutagenesis. Position of the mutations on the structure of IscS (PDB code: 3LVL) and HSQC-NMR spectra of 15N-labeled HscB 100 μM (black) and in the presence of the IscS mutants (1:1). Spectra in red are not affected, spectra in orange show the disappearance of few peaks because of the formation of a complex IscS:HscB. The same color coding is used in the structure panel.

Mentions: In turn, we tested the ability of ad hoc designed IscS mutants (IscS_R39E/W45E, IscS_K101E/K105E, IscS_R220E/R223E/R225E, IscS_I314E/M315E, and IscS_E334S/R340S) which affect different regions of the protein to identify the surface of IscS interacting with HscB. Circular dichroism supports that the mutants are all correctly folded as expected from their location in exposed regions of the enzyme (Prischi et al., 2010b). Among them, IscS_R220E/R223E/R225E, IscS_R39E/W45E, and IscS_K101E/K105E leave mostly unaffected the spectrum of 15N HscB (Figure 4). This means that mutations of these residues results in complete or partial abolishment of binding. Conversely, the spectrum is affected by IscS_I314E/M315E and IscS_E334S/R340S titration comparably to wild-type, indicating that these residues are not involved in the interaction. A similar behavior has been reported for the frataxin ortholog CyaY (Prischi et al., 2010b) and for Fdx (Yan et al., 2013) which also form complexes with IscS with micromolar affinities. These results thus suggest that HscB binds IscS in a site overlapping with that observed for these proteins.


A New Tessera into the Interactome of the isc Operon: A Novel Interaction between HscB and IscS
Identification of the surface of interaction on IscS by mutagenesis. Position of the mutations on the structure of IscS (PDB code: 3LVL) and HSQC-NMR spectra of 15N-labeled HscB 100 μM (black) and in the presence of the IscS mutants (1:1). Spectra in red are not affected, spectra in orange show the disappearance of few peaks because of the formation of a complex IscS:HscB. The same color coding is used in the structure panel.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Identification of the surface of interaction on IscS by mutagenesis. Position of the mutations on the structure of IscS (PDB code: 3LVL) and HSQC-NMR spectra of 15N-labeled HscB 100 μM (black) and in the presence of the IscS mutants (1:1). Spectra in red are not affected, spectra in orange show the disappearance of few peaks because of the formation of a complex IscS:HscB. The same color coding is used in the structure panel.
Mentions: In turn, we tested the ability of ad hoc designed IscS mutants (IscS_R39E/W45E, IscS_K101E/K105E, IscS_R220E/R223E/R225E, IscS_I314E/M315E, and IscS_E334S/R340S) which affect different regions of the protein to identify the surface of IscS interacting with HscB. Circular dichroism supports that the mutants are all correctly folded as expected from their location in exposed regions of the enzyme (Prischi et al., 2010b). Among them, IscS_R220E/R223E/R225E, IscS_R39E/W45E, and IscS_K101E/K105E leave mostly unaffected the spectrum of 15N HscB (Figure 4). This means that mutations of these residues results in complete or partial abolishment of binding. Conversely, the spectrum is affected by IscS_I314E/M315E and IscS_E334S/R340S titration comparably to wild-type, indicating that these residues are not involved in the interaction. A similar behavior has been reported for the frataxin ortholog CyaY (Prischi et al., 2010b) and for Fdx (Yan et al., 2013) which also form complexes with IscS with micromolar affinities. These results thus suggest that HscB binds IscS in a site overlapping with that observed for these proteins.

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.