Transition metal ion activation of DNA binding by the diphtheria tox repressor requires the formation of stable homodimers.

Xu, Tao; Zeng, Hui; Murphy, John R.

doi:10.1073/pnas.92.15.6803

Cited by 49 publications

(37 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, this same sensitivity can complicate molecular interpretation of the observed fluorescence changes. In the case of IdeR and other members of the DtxR family, fluorescence emission is quenched in the metal bound form (16,21). This quenching could result from the bound metal, whereas dimer formation (13,21) and stabilization of the N-terminal domain (12) may increase or decrease the tryptophan fluorescence emission.…”

Section: Metal Binding In Ider Ismentioning

confidence: 99%

“…Trp 104 is located at the dimer interface and becomes shielded from the solvent in the dimer, whereas Trp 94 is located toward the top of the N-terminal domain and is solvent exposed in the holorepressor (Figure 1). Both tryptophans are close to the primary and ancillary metal binding sites and are expected to be quenched by transition metals upon coordination (16,21). Thus, we expected that fluorescence quenching would provide a sensitive and quantitative measure of metal binding.…”

Section: Dimerization Energetics In Idermentioning

confidence: 99%

“…Tryptophan fluorescence was monitored from 290 to 450 nm, with excitation at 280 nm at room temperature (12,16,17). Apo-protein was titrated with 1-2 µL aliquots from 1 or 10 mM Ni(II)Cl 2 solutions.…”

Section: Construction Of Expression Vectorsmentioning

confidence: 99%

See 2 more Smart Citations

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

2006

View full text Add to dashboard Cite

The iron-dependent regulator (IdeR) is a 230-amino acid transcriptional repressor that regulates iron homeostasis, oxidative stress response and virulence in Mycobacterium tuberculosis. The natural ligand for IdeR is Fe(II), but Ni(II), Co(II), Cd(II), Mn(II), and Zn(II) also bind to and activate the protein in vitro. Protein activation by metal is a complex process involving metal-induced folding of the N-terminal domain, changes in the interaction between the N-and C-terminal domains, and the formation of homodimers. Here, we investigate the energetics of dimerization and metal binding in IdeR. The dimerization energetics were determined as a function of metal binding using equilibrium analytical ultracentrifugation. The equilibrium dimer dissociation constant of apo-IdeR was 4.0 µM at 20°C. The dissociation constant decreased to 0.5 µM in the presence of one equivalent of Ni(II)Cl 2 and decreased further (K d , 50 nM) in the presence of excess Ni(II). IdeR contains two tryptophan residues. The addition of Ni(II) induced changes in fluorescence intensity and emission maximum of the tryptophan residues that strongly depended on protein concentration. At low IdeR concentration, fluorescence was enhanced at low metal-to-protein ratios but was quenched at high metal-to-protein ratios. At high IdeR concentration, metal binding resulted only in fluorescence quenching. The fluorescence enhancement at low protein concentrations was buffer-dependent and required the presence of both tryptophans. Metal binding affinity was measured quantitatively using equilibrium dialysis. The results showed strongly positive cooperative binding of three equivalents of metal per monomer with an average apparent dissociation constant of 2.2 ( 0.3 µM and a Hill coefficient of 2. Metal binding was not cooperative in an IdeR variant that showed reduced affinity for dimer formation. The results of this study establish the positive cooperative nature of metal binding by IdeR and suggest that dimerization is a major contributor to cooperative binding. The strong coupling between metal binding and dimerization places specific constraints on the activation mechanism.Iron is an essential element for most living organisms, including bacteria, where it plays a critical role in metabolism, proliferation, and infection. IdeR 1 facilitates the adaptation of Mycobacteria to iron limitation encountered by the pathogen in the host cell. IdeR is a dual functional regulator that acts under iron-rich conditions as a repressor of siderophore biosynthesis genes (mbtA, mbtB, mbtI) and as an activator of iron storage (bfrA, bfrB) (1) and oxidative stress response (katG, sodA) (2, 3) genes. In addition, IdeR controls genes encoding proteins involved in general metabolism and virulence determinants (4). The diversity of cellular processes controlled by IdeR requires the finely tuned sensing of available iron levels, and IdeR appears to be the central element in this complex network. Null mutations in the ideR gene are lethal (5), further highlighting i...

show abstract

Section: Metal Binding In Ider Ismentioning

confidence: 99%

Section: Dimerization Energetics In Idermentioning

confidence: 99%

See 1 more Smart Citation

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

2006

View full text Add to dashboard Cite

show abstract

“…However, solution studies describe a more complex set of behaviors. ApoDtxR exists as a monomer in dilute solutions 12,13 and only forms a stable dimer after both metals have bound, though the sequence of metal ion binding is in dispute. 14,15 In the absence of bound metals, the first 124 residues of DtxR, spanning the DNA-binding and dimerization domain, experience significant flexibility, perhaps sampling multiple conformations.…”

Section: The Activation Of Mntrmentioning

confidence: 99%

“…[7][8][9][10] Because of the importance of these regulatory proteins in bacterial physiology and virulence, the mechanism of their action is of interest. In the past several years, the structural changes that relate to the activation of DtxR have received considerable attention, [11][12][13][14][15][16] but little is known about how its distantly related structural homolog, MntR, is activated by manganese binding. 2+ or Cd 2+ , another strongly activating metal ion.…”

mentioning

confidence: 99%

The Conformations of the Manganese Transport Regulator of Bacillus subtilis in its Metal-free State

Dewitt

Kliegman

Helmann

et al. 2007

Journal of Molecular Biology

View full text Add to dashboard Cite

The manganese transport regulator (MntR) from Bacillus subtilis binds cognate DNA sequences in response to elevated manganese concentrations. MntR functions as a homodimer that binds two manganese ions per subunit. Metal binding takes place at the interface of the two domains that comprise each MntR subunit: an N-terminal DNA-binding domain and a C-terminal dimerization domain. In order to elucidate the link between metal binding and activation, a crystallographic study of MntR in its metal-free state has been undertaken. Here we describe the structures of the native protein and a selenomethionine-containing variant, solved to 2.8 Å. The two structures contain five crystallographically unique subunits of MntR, providing diverse views of the metal-free protein. In apo-MntR, as in the manganese complex, the dimer is formed by dyad-related C-terminal domains that provide a conserved structural core. Similarly, each DNA-binding domain largely retains the folded conformation found in metal bound forms of MntR. However, compared to metal-activated MntR, the DNA-binding domains move substantially with respect to the dimer interface in apo-MntR. Overlays of multiple apo-MntR structures indicate that there is a greater range of positioning allowed between N and C-terminal domains in the metal-free state and that the DNA-binding domains of the dimer are farther apart than in the activated complex. To further investigate the conformation of the DNA-binding domain of apo-MntR, a site-directed spin labeling experiment was performed on a mutant of MntR containing cysteine at residue 6. Consistent with the crystallographic results, EPR spectra of the spin-labeled mutant indicate that tertiary structure is conserved in the presence or absence of bound metals, though slightly greater flexibility is present in inactive forms of MntR. KeywordsX-ray structure; transcriptional regulator; metal homeostasis; allosteryThe manganese transport regulator (MntR) controls manganese homeostasis in Bacillus subtilis. 1 When cellular levels of manganese are high, MntR represses the transcription of genes encoding manganese uptake transporters by binding to cognate operator sequences. [1][2][3] Manganese is an essential nutrient in bacteria and plays an important role in cellular defense 17,18 MntR is a functional homodimer of 142-residue subunits, each composed of two domains. The 71-residue N-terminal DNA-binding domain consists of three α-helices and two strands of antiparallel β-sheet (Figure 1), the latter forming the flexible "wing" of a winged helix-turn-helix (HTH) motif that interacts with DNA.17 , 19 The Cterminal domain contains four α-helices and forms the dimerization interface with its dyadrelated mate. The N and C-terminal domains are connected by a long linker helix (α4; residues 64-86) that extends from the wing to the dimer interface. Two manganese ions bind at the interface of the two domains in a single subunit, forming a binuclear complex that involves residues from the DNA-binding domain (Asp8 and Glu11), the dime...

show abstract

Diphtheria Toxin Repressor: Metal Ion Mediated Control of Transcription

Ringe

White

Chen

et al. 2004

Handbook of Metalloproteins

View full text Add to dashboard Cite

The diphtheria toxin repressor is a metal ion–activated DNA‐binding protein that prevents transcription of the diphtheria toxin gene. In its active form the repressor interacts with a semi‐palindromic operator sequence as two noninteracting dimers that recognize overlapping sequences. Recognition of the sequence occurs through two base‐specific interactions. The dimer binds two metal ions per monomer in its active form, only one of which is essential for DNA binding. The mechanism by which activation occurs includes the unraveling of the first turn of the N‐terminal helix in an indirect interaction with the critical metal ion that allows the repressor to interact optimally with the operator sequence of the toxin gene.

show abstract

Transition metal ion activation of DNA binding by the diphtheria tox repressor requires the formation of stable homodimers.

Cited by 49 publications

References 22 publications

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

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

The Conformations of the Manganese Transport Regulator of Bacillus subtilis in its Metal-free State

Diphtheria Toxin Repressor: Metal Ion Mediated Control of Transcription

Contact Info

Product

Resources

About