Characterization of the Iron Dependent Regulator and Its Hyperactive Mutant from Mycobacterium Tuberculosis

• Other Authors: Walker, Lawrence (authoraut)
• Format: Others
• Language: English; English
• Published: Florida State University
• Subjects: Chemistry; Biochemistry
• Online Access: http://purl.flvc.org/fsu/fd/FSU_migr_etd-8061

The Iron-dependent Regulator (IdeR) is a 230-amino acid transcriptional repressor that regulates iron homeostasis, oxidative stress response and virulence in Mycobacterium tuberculosis. IdeR binds to DNA as a dimer of dimers to repress gene expression and as four dimers to upregulate gene expression (upregulation is only for iron storage genes). IdeR contains two metal binding sites both of which have always been assumed to bind Fe(II) in vivo and it is the binding of metal by IdeR which induces the dimerization. IdeR is able to be activated for DNA binding in vitro by various divalent transition metals including: Mn(II), Fe(II), Co(II), Ni(II), Zn(II) and Cd(II). The model for activation of IdeR has always been investigated using divalent metals other than Fe(II) because of the rapid oxidation of Fe(II) to Fe(III). The objective of our work was to use Fe(II) to investigate the metal activation and DNA binding properties of IdeR. We also wanted to characterize a hyperactive mutant of IdeR (IdeR(D177K)) to better understand the cause of the hyperactive phenotype. To investigate the Fe(II) binding of IdeR we directly measured the Fe(II) bound using equilibrium dialysis and we investigated DNA binding using a fluorescence anisotropy assay. We determined that IdeR has the ability to bind Fe(II) into both metal binding sites, but that both sites bind the Fe(II) with affinities that differ by a factor of 25. We also determined that IdeR with only one Fe(II) bound per monomer was inactive and not able to bind to DNA. Through the use of IdeR metal binding site mutants we were able to determine that the first equivalent of Fe(II) bound to the ancillary metal site. This result was in contrast to all other papers published on IdeR, or its closest homologue (DtxR), that have shown that mutations to the primary metal binding site completely knock out repressor activity and mutations to the ancillary metal binding site only abrogate repressor activity. We determined that IdeR activation for promoter binding is enhanced when either Mn(II) or Zn(II) is present in solution before titrating in Fe(II), with Zn(II) having a much greater enhancement compared to Mn(II). We also determined that Zn(II) is able to bind at both metal binding sites in IdeR, but with KD values that differ by a factor of 70 and that the first equivalent of Fe(II) and the first equivalent of Zn(II) bind to different metal binding sites and the first equivalent of Zn(II) binding is ~36 picomolar. We determined that having Zn(II) present in solution before titrating in Fe(II) reduced the amount of Fe(II) required to activate promoter binding by 30 fold. These results suggest that IdeR is a mixed-metal repressor, where Zn(II) acts as a structural metal and Fe(II) acts to trigger the physiologically relevant promoter binding. We also set out to characterize the hyperactive repressor (IdeR(D177K)). We determined that this hyperactive mutant binds Fe(II) with a lower affinity that wild-type. The Fe(II) dependent promoter binding affinity is lower for IdeR(D177K) than wild-type IdeR. When Zn(II) is present we see that IdeR(D177K) also requires 30 fold less Fe(II) to activate promoter binding, but that IdeR(D177K) still requires a higher concentration of Fe(II) than wild-type IdeR. We also looked at the promoter binding affinity while keeping the metal concentrations constant and we saw that regardless of the metal species present (Fe(II), Zn(II), or Fe(II)-Zn(II)) IdeR(D177K) and wild-type IdeR had similar affinities for the promoter. IdeR(D177K) does not have a decrease sensitivity to changes in the free metal concentration once bound to the promoter as evidenced by our EDTA titration assay. The hyperactivity of IdeR(D177K) is due to it being bound to the promoter for a longer period of time compared to wild-type IdeR when both Zn(II) and Fe(II) are present. === A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy. === Summer Semester, 2013. === June 28, 2013. === DNA, Iron, Mycobacterium tuberculosis, Promoter region, Repressor, Zinc === Includes bibliographical references. === Timothy M. Logan, Professor Directing Dissertation; Michael Blaber, University Representative; Brian Miller, Committee Member; Lei Zhu, Committee Member.