Iron-Sulfur cofactor mediated recognition of two classes of binding sites by the Escherichia coli transcription factor IscR

IscR from Escherichia coli is an unusual Fe-S transcription factor, in that the [2Fe-2S]-form binds two classes of DNA sites (type 1 and 2), whereas the clusterless (apo) form binds only a single set of sites (type 2). Here, we report structural and biochemical studies of IscR with its DNA binding sites, which suggest that remodeling of the protein-DNA interface upon ligation of a [2Fe-2S] cluster cofactor broadens the DNA binding specificity from binding only a type 2 motif to both type 1 and type 2 motifs. We analyzed the sequence requirements for [2Fe-2S]-IscR binding of the type 1 motif, which revealed a conserved asymmetrical discriminator sequence element which is required for type 1 DNA site binding specificity. In contrast, structure-guided mutagenesis studies indicated that apo-IscR formed symmetrical interactions in both half sites of the symmetrical type 2 motif, suggesting that the asymmetrical nature of the type 1 site may exclude apo-IscR from binding these sites. Analysis of an apo-IscR variant with relaxed target-site discrimination identified a key residue in wild-type apo-IscR that we propose makes unfavorable interactions with one half site of the asymmetrical type 1 motif. Upon ligation of a [2Fe-2S]-cluster, these interactions appear to be removed, thereby allowing [2Fe-2S]-IscR to bind both type 1 and 2 motifs. These data suggest a novel mechanism of ligand-mediated DNA site recognition, whereby metallocluster ligation increases conformational flexibility in the DNA binding domain to permit relocation of a protein specificity determinant. Thus, [2Fe-2S]-IscR has increased DNA target site selection than apo-IscR, which allows a broader transcriptomic response by the [2Fe-2S]-form of the regulator.