Diphtheria toxin repressor (DtxR) regulates the expression of iron-sensitive genes in Corynebacterium diphtheriae, including the diphtheria toxin gene. DtxR contains an N-terminal metal- and DNA-binding domain that is connected by a proline-rich flexible peptide segment (Pr) to a C-terminal src homology 3 (SH3)-like domain. We determined the solution structure of the intramolecular complex formed between the proline-rich segment and the SH3-like domain by use of NMR spectroscopy. The structure of the intramolecularly bound Pr segment differs from that seen in eukaryotic prolylpeptide-SH3 domain complexes. The prolylpeptide ligand is bound by the SH3-like domain in a deep crevice lined by aliphatic amino acid residues and passes through the binding site twice but does not adopt a polyprolyl type-II helix. NMR studies indicate that this intramolecular complex is present in the apo-state of the repressor. Isothermal equilibrium denaturation studies show that intramolecular complex formation contributes to the stability of the apo-repressor. The binding affinity of synthetic peptides to the SH3-like domain was determined using isothermal titration calorimetry. From the structure and the binding energies, we calculated the enhancement in binding energy for the intramolecular reaction and compared it to the energetics of dimerization. Together, the structural and biophysical studies suggest that the proline-rich peptide segment of DtxR functions as a switch that modulates the activation of repressor activity.
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.
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