Characterization of metal linked activation of the iron-dependent regulator from Mycobacterium tuberculosis

The Iron-dependent regulator (IdeR) protein is a member of the diphtheria toxin repressor (DtxR) family of metal responsive proteins that regulates genes involved in iron homeostasis, oxidative stress response, and virulence in Mycobacterium tuberculosis. The roles of metals involved in activating IdeR are not completely understood. A better understanding of the metal binding process could possibly provide insight to a metal's role in virulence and other cell functions. The biological activating metal for IdeR is Fe(II), but several divalent metals including Ni(II), Co(II), Zn(II), Mn(II), and Cd(II) have been shown to activate the protein in vitro. The current model for the activation process involves IdeR selectively binding Fe(II) from cytosolic pools of free iron and becoming competent to bind DNA by structural organization and formation of a homodimer. The metal selection and dimerization processes are not well understood. Two drawbacks from previous work on this system are the use of in vivo assays which lack the details of metal binding and the absence of in vitro studies using Fe(II) as the metal ligand. This work provides a better understanding of the selection process of Fe(II) versus other metals as well as the dimerization effects of different metals.;Thermodynamics of metal selection by Fe(II) and other metals was investigated using equilibrium dialysis. The results showed that Zn(II) and Fe(II) bound two equivalents of metal per monomer with each equivalent's affinity differing by 65-fold and 24-fold, respectively. Site mutant studies clearly showed Fe(II) bound to the ancillary site with high affinity while the Zn(II) results were less clear, but suggested that Zn(II) bound the primary site with high affinity. The equilibrium assays was modified to determine binding of more than one metal species. The mixed metal binding studies clearly showed that IdeR bound 1 equivalent each of Zn(II) and Fe(II). The fxbA promoter binding assay with 1 equivalent of Zn(II) and Fe(II) bound exhibited 30-fold tighter DNA binding than with Fe(II) alone. While the Fe(II) alone affinity is fairly weak, the enhanced affinity is in the range of most DNA binding regulators from other systems.;Dimerization is important in the function of most DNA binding regulators. The enhancement of the dimerization of IdeR due to metal binding was studied using Analytical Ultracentrifugation. The sedimentation velocity analysis revealed a larger fraction of dimer present in IdeR samples when Zn(II) was bound versus Fe(II).;The metal binding results of both Fe(II) and Zn(II) is novel to this family of repressor proteins which lead to questions about the role of each metal in IdeR. The binding processes of Fe(II) and Zn(II) were studied using Molecular Dynamic simulations. The free energies of Fe(II) and Zn(II) were computed using the CHARMM software and compared for relative differences in affinity at both sites between monomer and dimer configurations. The analysis of the trajectory data concluded that there are small subtle structural changes that are initiated by Zn(II) binding leading to dimerization.;The information obtained in the studies shed new light on the metal binding selectivity of IdeR which was previously thought to only bind Fe(II). Zn(II) plays an important role in IdeR activation by enhancing DNA binding by stabilizing the dimer form.