title: Sequence of ligand binding and structure change in the diphtheria toxin repressor upon activation by divalent transition metals.
abstract: The diphtheria toxin repressor (DtxR) is an Fe(II)-activated transcriptional regulator of iron homeostatic and virulence genes in Corynebacterium diphtheriae. DtxR is a two-domain protein that contains two structurally and functionally distinct metal binding sites. Here, we investigate the molecular steps associated with activation by Ni(II)Cl(2) and Cd(II)Cl(2). Equilibrium binding energetics for Ni(II) were obtained from isothermal titration calorimetry, indicating apparent metal dissociation constants of 0.2 and 1.7 microM for two independent sites. The binding isotherms for Ni(II) and Cd(II) exhibited a characteristic exothermic-endothermic pattern that was used to infer the metal binding sequence by comparing the wild-type isotherm with those of several binding site mutants. These data were complemented by measuring the distance between specific backbone amide nitrogens and the first equivalent of metal through heteronuclear NMR relaxation measurements. Previous studies indicated that metal binding affects a disordered to ordered transition in the metal binding domain. The coupling between metal binding and structure change was investigated using near-UV circular dichroism spectroscopy. Together, the data show that the first equivalent of metal is bound by the primary metal binding site. This binding orients the DNA binding helices and begins to fold the N-terminal domain. Subsequent binding at the ancillary site completes the folding of this domain and formation of the dimer interface. This model is used to explain the behavior of several mutants.
authors.affiliation: Kasha Laboratory, Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306-4380, USA.
authors.forename: Vijayaraghavan
authors.initials: V
authors.lastname: Rangachari
authors.forename: Vedrana
authors.initials: V
authors.lastname: Marin
authors.forename: Ewa A
authors.initials: EA
authors.lastname: Bienkiewicz
authors.forename: Maria
authors.initials: M
authors.lastname: Semavina
authors.forename: Luis
authors.initials: L
authors.lastname: Guerrero
authors.forename: John F
authors.initials: JF
authors.lastname: Love
authors.forename: John R
authors.initials: JR
authors.lastname: Murphy
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Logan
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D002942:Circular Dichroism; D003353:Corynebacterium diphtheriae / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009532:Nickel / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D002413:Cations, Divalent; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C075586:DtxR protein, Corynebacterium diphtheriae; D008024:Ligands; D011994:Recombinant Proteins; D002104:Cadmium; D009532:Nickel