Solution structure and peptide binding studies of the C-terminal Src homology 3-like domain of the diphtheria toxin repressor protein

Abstract

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...

The src Homology 3-Like Domain of the Diphtheria Toxin Repressor (DtxR) Modulates Repressor Activation through Interaction with the Ancillary Metal Ion-Binding Site

“…In addition, it appears that certain residues in this domain (e.g., position 175) are able to modulate repressor activation by affecting behavior of at least the ancillary cation-binding site. Others have reported the SH3-like fold found in the C-terminal domain (14,26), as well as the ability of this isolated domain to bind in vitro a proline-rich peptide mimicking residues 125 to 139 of DtxR (26). The elucidation of the overall biological role of the Cterminal SH3-like domain in the activation of DtxR remains a focus of active research in many laboratories.…”

“…This domain appears to undergo the more significant structural change during metal ion-induced activation, changing from a molten globule in the apo form (25) to an organized structure upon cation binding. In contrast, the Cterminal domain (approximately residues 148 to 226) folds into an src homology 3 (SH3)-like structure (14,26). Although in vitro experiments have demonstrated a weak interaction between the purified C-terminal domain and a proline-rich peptide identical in sequence to residues 125 to 139 of DtxR, the biological function of this domain remains unclear (26).…”

“…In contrast, the Cterminal domain (approximately residues 148 to 226) folds into an src homology 3 (SH3)-like structure (14,26). Although in vitro experiments have demonstrated a weak interaction between the purified C-terminal domain and a proline-rich peptide identical in sequence to residues 125 to 139 of DtxR, the biological function of this domain remains unclear (26). Some crystallographic reports have suggested that the C-terminal domain donates two ligands to the ancillary cation-binding site, but no biological function of these residues has been reported to date (12).…”

Solution Structure of Escherichia coli FeoA and Its Potential Role in Bacterial Ferrous Iron Transport

“…5B). The presence of metal ions signals these proteins to transition between the DNA-binding and nonbinding forms (61)(62)(63)(64)(65)(66)(67)(68)(69)(70)73). The SH3-like domains of IdeR and DtxR also play an important role in this conversion apart from sole metal binding.…”

“…These results revealed similarity to streptococcal coaggregation regulator (ScaR) (PDB ID 3hru) (Z-score, 7.1), iron-dependent regulator (IdeR) (PDB ID 1u8r) (Z-score, 6.7), and diphtheria toxin repressor protein (DtxR) (PDB ID 1c0w) (Z-score, 5.6). These proteins are all part of the metal and DNA-binding protein families that function as transcriptional regulators (61)(62)(63)(64)(65)(66)(67)(68). In addition, these proteins all have three domains: a DNA-binding, a dimerization, and an SH3-like domain (64,69,70), where the structure of FeoA resembles the last domain (see Fig.…”