Biophysical characterization of the complex between the iron-responsive transcription factor Fep1 and DNA

Fep1 is an iron-responsive GATA-type transcriptional repressor present in numerous fungi. The DNA-binding domain of this protein is characterized by the presence of two zinc fingers of the Cys(2)-Cys(2) type and a Cys-X-5-Cys-X-8-Cys-X-2-Cys motif located between the two zinc fingers, that is involved in binding of a [2Fe-2S] cluster. In this work, biophysical characterization of the DNA-binding domain of Pichia pastoris Fep1 and of the complex of the protein with cognate DNA has been undertaken. The results obtained by analytical ultracentrifugation sedimentation velocity, small-angle X-ray scattering and differential scanning calorimetry indicate that Fep1 is a natively unstructured protein that is able to bind DNA forming 1:1 and 2:1 complexes more compact than the individual partners. Complex formation takes place independently of the presence of a stoichiometric [2Fe-2S] cluster, suggesting that the cluster may play a role in recruiting other protein(s) required for regulation of transcription in response to changes in intracellular iron levels.