Domain-Domain Interactions on the Activation Pathway of Diphtheria Toxin Repressor Protein (DTXR)

Diphtheria toxin repressor (DtxR) is an Fe(II)-activated transcriptional regulator of iron homeostatic and virulence genes in Corynebacterium diphtheriae and co-regulates expression of 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. Transition metal ions are bound in two structurally and functionally distinct sites, and metal binding effects a disordered-to-ordered structural transition, changes the interactions between the two domains, and increases the affinity for dimer formation. Here we investigate the inactive state of the repressor and domain-domain interactions that are present in metal-free DtxR. We follow that study by investigation of the changes in the domain-domain interactions upon metal activation of the repressor. Genetic analysis shows that the SH3 C-terminal domain regulates the activity of the N-terminal domain. Structural studies suggest two possible mechanisms for interactions between the two domains. Here we establish that the SH3 domain of DtxR is required for optimal repressor activity using in vivo repressor assays. Solution NMR structure of the C-terminal PrSH3 construct shows that the Pr segment connecting the two domains is bound by the SH3 domain in a deep crevice lined by hydrophobic amino acids and does not resemble the eukaryotic polyprolyl type-II helix. NMR studies presented here confirm that this intramolecular complex is present in the apo-repressor. Isothermal equilibrium denaturation studies show that the intramolecular complex contributes to the stability of the aporepressor. From the structural and thermodynamic data, we conclude that the proline-rich segment of DtxR functions as a switch that modulates the activation of the repressor. Our study of the metal activated repressor shows that the apparent metal dissociation constants are 0.2 and 1.7 mM for two independent sites. These data were complemented by measuring the distances between specific backbone amide nitrogens and the first equivalent of metal through heteronuclear NMR relaxation measurements. Previous studies indicate that metal binding affects a disordered to ordered transition in the metal binding domain. The coupling between metal binding and structure was investigated using near-UV circular dichroism and 19F NMR 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 the N domain and formation of the dimerization interface. This model is used to explain the behavior of several mutants.