Prolylpeptide Binding by the Prokaryotic SH3-like Domain of the Diphtheria Toxin Repressor:  A Regulatory Switch^,

Abstract

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.

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

“…The SH3-like domains of IdeR and DtxR also play an important role in this conversion apart from sole metal binding. They interact with the other domains of the protein either on an intermolecular or intramolecular level that results in global conformational changes, allowing the binding or release of DNA (62,63,(65)(66)(67)(68)74). Examining our solution NMR studies, done at high protein concentrations, has shown that FeoA is a monomeric protein, making it unlikely that it dimerizes noncovalently in a similar fashion to IdeR for function.…”

“…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.…”

“…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.…”

“…Examining our solution NMR studies, done at high protein concentrations, has shown that FeoA is a monomeric protein, making it unlikely that it dimerizes noncovalently in a similar fashion to IdeR for function. Of the three homologues, the SH3-like domain of ScaR is only known to bind metal, with no specific function like the other two (64,68). FeoA shares greater structural similarity between all three C-terminal domains of these proteins than with the prototypical SH3 domains ( Fig.…”

Mn(II) Binding by the Anthracis Repressor from Bacillus anthracis

“…Target DNA binding by metal-activated DtxR and IdeR is cooperative (28,29), as the binding of the first dimer enhances the binding of the second. Cooperative metal binding in AntR may be an evolutionary step compensating for the lack of a SH3-like domain, which has a regulatory role in DtxR (6).…”