Genetic and biophysical studies of diphtheria toxin repressor (DtxR) and the hyperactive mutant DtxR(E175K) support a multistep model of activation

Abstract

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...

Both Corynebacterium diphtheriae DtxR(E175K) and Mycobacterium tuberculosis IdeR(D177K) Are Dominant Positive Repressors of IdeR-Regulated Genes in M. tuberculosis

“…Although the C-terminal domain is less well conserved, crystallography has confirmed that the secondary structures forming the SH3-like fold (7) and its interaction with the ancillary metal ion binding site exist in both DtxR and IdeR (35). Nuclear magnetic resonance analysis has confirmed that the mutant DtxR(E175K) adopts a more ordered conformation through binding of the SH3-like domain to the polypropyl tether region between the N-and C-terminal domains (17).…”

Metal-Linked Dimerization in the Iron-Dependent Regulator from Mycobacterium tuberculosis

“…Initial crystal structures clearly indicated two metal binding sites per monomer in either protein (7,9,10,(24)(25)(26). The functional significance of these sites for repressor function have been thoroughly investigated in DtxR (9,(27)(28)(29). More recently, a third metal binding site located in the SH3 domain was identified in the crystal structure of IdeR (6,7), although the biological relevance of this site has not been established.…”

Transcription of the ContiguoussigB,dtxR, andgalEGenes inCorynebacterium diphtheriae: Evidence for Multiple Transcripts and Regulation by Environmental Factors

“…4C). Clone dtxRU1 produced 20 to 30 Miller units of ␤-galactosidase activity, and its P-dtxR promoter was not regulated by DtxR or iron. In control experiments, however, the P-tox promoter did show the expected pattern of DtxR-dependent repression by iron.…”

“…The final 86 amino acids of DtxR (residues 141 to 226) comprise domain 3, which contributes ligands to one of the metal binding sites and has topology similar to the SH3 domains found in some eukaryotic signal transduction proteins (44). The first two domains of DtxR are essential for DtxR to function as a transcriptional repressor, while domain 3 is thought to modulate the activity of DtxR at individual promoters (30,41).…”