The transcription factor NF-κB interacts with many other co-regulator proteins that modulate its binding and transcriptional activity. One of these co-regulators, Pirin, is an iron-dependent metalloprotein that has been shown to enhance the DNA binding of NF-κB homodimers. Here, we characterize the interactions between Pirin and its known NF-κB binding partners and examined the role of Bcl-3, a protein that is required for Pirin’s interaction with p50. In addition, we use site-directed mutagenesis to alter conserved residues within Pirin’s metal binding environment and observed how it affected the DNA binding and conformation of the Pirin-NF-κB complex. These studies show that, while a similar enhancing effect on DNA binding is observed, the interactions of Pirin with different NF-κB members are distinct from each other and could possibly have different physiological purposes.

The NF-κB family of transcription factors controls a number of essential cellular functions. Pirin is a non-heme iron (Fe) redox specific co-regulator of NF-κB (p65) and has been shown to modulate the affinity between the homodimeric p65 and the DNA. The allosteric effect of the active Fe(III) form of Pirin on the DNA is not known and has not been investigated. We carry out multiple microsecond-long molecular dynamics simulations of the free DNA, p65-DNA complex, and the Pirin-p65-DNA supramolecular complexes in explicit water. We show that, unlike the Fe(II) form of Pirin, the Fe(III) form in the Pirin-p65-DNA supramolecular complex enhances the interactions and affinity between p65 and the DNA, in agreement with experiments. The results further provide atomistic details of the effect of the Fe(III) form of Pirin on the DNA upon binding to p65 to form the supramolecular complex.

Reisebericht über die Durchquerung des Rila- und Pirin-Gebirges in Bulgarien