The diphtheria toxin repressor (DtxR) is the best-characterized member of a family of homologous proteins that regulate iron uptake and virulence gene expression in the Gram-positive bacteria. DtxR contains two domains that are separated by a short, unstructured linker. The N-terminal domain is structurally well-defined and is responsible for Fe 2؉ binding, dimerization, and DNA binding. The C-terminal domain adopts a fold similar to eukaryotic Src homology 3 domains, but the functional role of the C-terminal domain in repressor activity is unknown. The solution structure of the C-terminal domain, consisting of residues N130-L226 plus a 13-residue N-terminal extension, has been determined by using NMR spectroscopy. Residues before A147 are highly mobile and adopt a random coil conformation, but residues A147-L226 form a single structured domain consisting of five -strands and three helices arranged into a partially orthogonal, two-sheet -barrel, similar to the structure observed in the crystalline Co 2؉ complex of full-length DtxR. Chemical shift perturbation studies demonstrate that a proline-rich peptide corresponding to residues R125-G139 of intact DtxR binds to the C-terminal domain in a pocket formed by residues in -strands 2, 3, and 5, and helix 3. Binding of the prolinerich peptide by the C-terminal domain of DtxR presents an example of peptide binding by a prokaryotic Src homology 3-like protein. The results of this study, combined with previous x-ray studies of intact DtxR, provide insights into a possible biological function of the C-terminal domain in regulating repressor activity.The diphtheria toxin repressor (DtxR) regulates the expression of several iron-sensitive genes in Corynebacterium diphtheriae, including the iron-response protein genes (irp), a heme oxygenase, and the diphtheria toxin gene (tox) (1-3). DtxR is the best-characterized member of a family of homologous proteins that regulate iron uptake and virulence gene expression in the Gram-positive bacteria, including Mycobacterium tuberculosis, Brevibacterium lactofermentum, Streptomyces lividans, and Streptomyces pilosus (4, 5). The N-terminal 136 aa of this 226-aa protein form a DNA-binding dimer, whose structure has been determined by x-ray crystallography with (6-10) and without (7, 10) divalent cations, and while bound to a DNA operator sequence (11). The structure of the C-terminal 90 aa, which is more mobile than the N-terminal domain in all crystal forms, has been mapped in the Co 2ϩ -DtxR complex (9) and shown to be structurally homologous to eukaryotic Src homology 3 (SH3) domains, although the amino acid sequence homology is low. A limited number of mutations in the C-terminal domain (12) or the linker region (13) affect repressor activity, but the structural nature of this regulation by the C-terminal domain has been obscure.To obtain a more complete understanding of the function of the intact repressor protein, we are focusing on the C-terminal domain. From the sequential assignment of resonances in heteronuclear N...
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.
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