Abstract
The Iron-dependent Regulator (IdeR) is a 230-amino acid transcriptional repressor that regulates iron homeostasis, oxidative stress response and virulence in Mycobacterium tuberculosis. The importance of IdeR for mycobacterial metabolism, virulence, and survival in the host cell suggests that the protein may have a potential as a therapeutic target. The natural ligand for IdeR is Fe(II), but Ni(II), Co(II), Cd(II), Mn(II), and Zn(II) also bind to and activate the protein in vitro. Protein activation by metal is complex process involving metal-induced folding of the N-terminal domain, changes in the interaction between the N- and C-terminal domains, and formation of homodimers. The objective of this work was to characterize the molecular details of IdeR activation in vitro. The dimerization energetics were determined as a function of metal binding using equilibrium analytical ultracentrifugation. The dissociation constant was strongly dependent on the metal ligation state of the protein. Addition of Ni(II) induced changes in fluorescence intensity and emission maximum of the tryptophan residues that strongly depended on protein concentration. Mutational analysis suggested that both tryptophan residues in IdeR are sensitive to folding, dimerization and metal binding. Metal binding affinity was measured quantitatively using equilibrium dialysis. The results showed
strongly positive cooperative binding of three equivalents of metal per monomer. Metal binding was not cooperative in an IdeR variant that showed reduced affinity for dimer formation. The results of this study establish the positive cooperative nature of metal binding by IdeR and suggest that dimerization contributes significantly to the cooperative binding. Equilibrium binding studies together with fluorescence titration and isothermal titration calorimetry experiments performed in metal binding mutants revealed equal importance of the primary and ancillary metal binding sites for cooperativity of metal binding, in contrast to the differential role of each site in repressor activity in vitro. The strong coupling between metal binding and dimerization establishes “all or nothing” mechanism for regulation of repressor activity under the conditions of constantly changing free metal concentration, providing greater control over the metal-dependent DNA binding activity of IdeR. Our findings place specific constraints on the activation mechanism, simplifying the existing model.
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