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.

The diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae is the prototypic member of a superfamily of transition metal ion-activated transcriptional regulators that have been isolated from Gram-positive prokaryotes. Upon binding divalent transition metal ions, the N-terminal domain of DtxR undergoes a dynamic structural organization leading to homodimerization and target DNA binding. We have used site-directed mutagenesis and NMR analysis to probe the mechanism by which apo-DtxR transits from an inactive to a fully active repressor upon metal ion binding. We demonstrate that the ancillary metal-binding site mutant DtxR(H79A) requires higher concentrations of metal ions for activation both in vivo and in vitro, providing a functional correlation to the proposed cooperativity between ancillary and primary binding sites. We also demonstrate that the C-terminal src homology 3 (SH3)-like domain of DtxR functions to modulate repressor activity by (i) binding to the polyprolyl tether region between the N-and C-terminal domains, and (ii) destabilizing the ancillary binding site, leading to full inactivation of the repressor. Finally, we show by NMR analysis that the hyperactive phenotype of DtxR(E175K) results from the stabilization of a structural intermediate in the activation process. Taken together, the data presented support a multistep model for the activation of apo-DtxR by transition metal ions. In Corynebacterium diphtheriae, the diphtheria toxin repressor (DtxR) is an iron-sensitive transcriptional regulator that controls the expression of a number of genes, including the diphtheria toxin structural gene (tox), a number of operons involved in iron uptake and homeostasis, and outer surface lipoproteins (1-4). DtxR is the prototypic member of a large family of transition metal ion-activated repressors in Gram-positive prokaryotes (5). In the absence of divalent transition metal ions, apo-DtxR exists as an inactive monomer that is in weak equilibrium with a dimeric form (6). Whereas Fe 2ϩ is the physiological activator of DtxR in vivo, Fe 2ϩ , Ni , and Mn 2ϩ have been shown to activate the aporepressor in vitro (7). Once activated, DtxR forms stable dimers, and two pairs of dimers have been shown to bind to almost opposite faces of the tox operator (toxO) sequence (6,8).DtxR is a 226-aa 26-kDa protein composed of two major structural domains linked by a 23-aa flexible tether that contains a proline-rich region (1, 9, 10). The N-terminal domain (residues 1-123) contains the ancillary and primary metal ion-binding sites, a canonical helix-turn-helix DNA-recognition motif, and an extensive hydrophobic surface necessary for the formation of stable dimers (9-11). We have previously shown that alanine substitution of any of the primary site residues (Met-10, Cys-102, Glu-105, and His-106) results in complete inactivation of repressor activity, whereas alanine substitution of any of the ancillary site residues (His-79, Glu-83, His-98, Glu-170, and Gln-173) results in a partial loss of activity (11,12). Bec...

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Previous studies demonstrated that intra‐domain interactions between Src family kinases (SFKs), stabilized by binding of the phosphorylated C‐terminus to the SH2 domain and/or binding of the SH2 kinase linker to the SH3 domain, lock the molecules in a closed conformation, disrupt the kinase active site, and inactivate SFKs. Here we report that the up‐regulation of N‐methyl‐d‐aspartate receptors (NMDARs) induced by expression of constitutively active neuronal Src (n‐Src), in which the C‐terminus tyrosine is mutated to phenylalanine (n‐Src/Y535F), is significantly reduced by dysfunctions of the SH2 and/or SH3 domains of the protein. Furthermore, we found that dysfunctions of SH2 and/or SH3 domains reduce auto‐phosphorylation of the kinase activation loop, depress kinase activity, and decrease NMDAR phosphorylation. The SH2 domain plays a greater regulatory role than the SH3 domain. Our data also show that n‐Src binds directly to the C‐terminus of the NMDAR NR2A subunit in vitro, with a KD of 108.2 ± 13.3 nm. This binding is not Src kinase activity‐dependent, and dysfunctions of the SH2 and/or SH3 domains do not significantly affect the binding. These data indicate that the SH2 and SH3 domains may function to promote the catalytic activity of active n‐Src, which is important in the regulation of NMDAR functions. Structured digital abstract  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8074560: NR2A (uniprotkb:http://www.uniprot.org/uniprot/Q00959) binds (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) to n‐Src (uniprotkb:http://www.uniprot.org/uniprot/P05480) by surface plasmon resonance (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0107)  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8074641, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8074668, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8074679, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8074693, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8074813: n‐Src (uniprotkb:http://www.uniprot.org/uniprot/P05480) and n‐Src (uniprotkb:http://www.uniprot.org/uniprot/P05480) phosphorylate (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0217) by protein kinase assay (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0424)  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8074576, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8074726, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8074741, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8074777: n‐Src (uniprotkb:http://www.uniprot.org/uniprot/P05480) phosphorylates (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0217) NR2A (uniprotkb:http://www.uniprot.org/uniprot/Q00959) by protein kinase assay (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0424)

Summary Neuronal Src (n-Src) is an alternative isoform of Src kinase containing a 6-amino acid insert in the SH3 domain that is highly expressed in neurons of the central nervous system (CNS). To investigate the function of n-Src, wild-type n-Src, constitutively active n-Src in which the C-tail tyrosine 535 was mutated to phenylalanine (n-Src/Y535F) and inactive n-Src in which the lysine 303 was mutated to arginine in addition to the mutation of Y535F (n-Src/K303R/Y535F), were expressed and purified from E. coli BL21(DE3) cells. We found that all three types of n-Src constructs expressed at very high yields (~500 mg/L) at 37°C, but formed inclusion bodies. In the presence of 8 M urea these proteins could be solubilized, purified under denaturing conditions, and subsequently refolded in the presence of arginine (0.5 M). These Src proteins were enzymatically active except for the n-Src/K303R/Y535F mutant. n-Src proteins expressed at 18°C were soluble, albeit at lower yields (~10 – 20 mg/L). The lowest yields were for n-Src/Y535F (~10 mg/L) and the highest for n-Src/K303R/Y535F (~20 mg/L). We characterized the purified n-Src proteins expressed at 18°C. We found that altering n-Src enzyme activity either pharmacologically (e.g., application of ATP or a Src inhibitor) or genetically (mutation of Y535 or K303) was consistently associated with changes in n-Src stability: an increase in n-Src activity was coupled with a decrease in n-Src stability and vice versa. These findings, therefore, indicate that n-Src activity and stability are interdependent. Finally, the successful production of functionally active n-Src in this study indicates that the bacterial expression system may be a useful protein source in future investigations of n-Src regulation and function.

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