Sequence of Ligand Binding and Structure Change in the Diphtheria Toxin Repressor upon Activation by Divalent Transition Metals

Rangachari, Vijayaraghavan; Marin, Vedrana; Bienkiewicz, Ewa A.; Semavina, Maria; Guerrero, Luis A. Chel; Love, J.; Murphy, John R.; Logan, Timothy M.

doi:10.1021/bi047825w

Cited by 23 publications

(37 citation statements)

References 41 publications

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“…Rangachari et al (30) confirmed that DtxR bound more than 2 equivalents of Ni(II) per monomer and that the binding appeared to be cooperative. Chou et al were the first to report positive cooperative metal binding for IdeR (16).…”

Section: Metal Binding In Ider Ismentioning

confidence: 96%

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

2006

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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...

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Section: Metal Binding In Ider Ismentioning

confidence: 96%

“…However, a recent high-resolution structure of IdeR (6,7) and metal binding studies of DtxR (8) are consistent with more than two equivalents of metal bound per monomer. One metal binding site is formed entirely by the residues from the N-terminal domain (M10, C102, E105, and H106) ( Figure 1).…”

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confidence: 89%

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

2006

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“…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. 16 Upon metal binding, dimers form and the structure becomes ordered.…”

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.…”

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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

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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...

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“…metalloregulator has become a prominent subject of investigation (4)(5)(6)(7)(8)(9)(10). Indeed, many metalloregulators are reported to bind several metal ions in vitro, while only eliciting a specific transcriptional response when they are bound to the cognate metal in vivo (5)(6)(7)9).…”

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Metal Binding Studies and EPR Spectroscopy of the Manganese Transport Regulator MntR

et al. 2006

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Manganese transport regulator (MntR) is a member of the diphtheria toxin repressor (DtxR) family of transcription factors that is responsible for manganese homeostasis in [4295][4296][4297][4298][4299][4300][4301][4302][4303], and generally follow the Irving-Williams series. Direct detection of the dinuclear Mn 2+ site in MntR with EPR spectroscopy is presented, and the exchange interaction was determined, J = -0.2 cm -1 . This value is lower in magnitude than most known dinuclear Mn 2+ sites in proteins and synthetic complexes and is consistent with a dinuclear Mn 2+ site with a longer Mn···Mn distance (4.4 Å) observed in some of the available crystal structures. MntR is found to have a surprisingly low binding affinity (∼160 μM) for its cognate metal ion Mn 2+ . Moreover, the results of DNA binding studies in the presence of limiting metal ion concentrations were found to be consistent with the measured metal-binding constants. The metalbinding affinities of MntR reported here help to elucidate the regulatory mechanism of this metaldependent transcription factor.Bacteria handle the delicate issue of metal ion homeostasis using a class of transcription factors known as metalloregulatory proteins (metalloregulators). In some systems, these metal-sensing proteins are involved in mediating the removal of toxic metals, while in other systems they are central to maintaining the required levels of essential metals. To date, a large number of metalloregulatory proteins have been identified that respond to a variety of metal ions (1-3). The subject of how metal binding is translated into an ability to control transcription via a † This work was supported by the University of California, San Diego, the Hellman Family fund, a Cottrell Scholar Award from the Research Corporation (S.M.C.), and the National Institutes of Health GM077387 (M.P.H.). * Author to whom correspondence should be adddressed. (M.P.H.) Telephone: (412) 268-1058; fax: (412) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript metalloregulator has become a prominent subject of investigation (4-10). Indeed, many metalloregulators are reported to bind several metal ions in vitro, while only eliciting a specific transcriptional response when they are bound to the cognate metal in vivo (5)(6)(7)9). This observation naturally leads to the question: how does a metalloregulator selectively respond to its cognate metal ion as opposed to other available metal ion activators? The ability of a metalloregulator to respond selectively to a metal ion may depend on several factors, including the availability of the requisite metal ion, the binding affinity for the metal ion, the charge on the metal ion, and the coordination geometry/number assumed by the metal ion upon binding. Determining which of these factors are most important for a given metalloregulator is essential for gaining a better understanding of how these proteins elicit transcriptional control.The manganese transport regulator MntR 1 is found in Bacillus subtilis and is ...

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Sequence of Ligand Binding and Structure Change in the Diphtheria Toxin Repressor upon Activation by Divalent Transition Metals

Cited by 23 publications

References 41 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

Metal Binding Studies and EPR Spectroscopy of the Manganese Transport Regulator MntR

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