Structure of the manganese-bound manganese transport regulator of Bacillus subtilis

Glasfeld, Arthur; Guédon, Emmanuel; Helmann, John D.; Brennan, Richard G.

doi:10.1038/nsb951

Cited by 92 publications

(141 citation statements)

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“…We cannot conclude from the DEER spectra whether the width in distance distribution is due to motions inherent in the spinlabel on AntR, multiple conformers of the backbone, or both. However, the intermonomer C R -C R distance for the homologous residue (M15) in MntR is 4.1 nm (12). Therefore, considering the extension of the spin-label side chains, the observed distance between nitroxides is in agreement with this structure.…”

Section: Discussionsupporting

confidence: 66%

“…In these proteins, which include TroR from Treponema pallidum (7), MntR from Bacillus subtilis (8,9), MDR1 from Archeolobus fulgidis (10), and AntR from Bacillus anthracis (11), the DNA binding motif and the residues forming the two metal ion binding sites are strongly conserved with the two-domain members of the DtxR family, but the C-terminal part of the protein is truncated, lacking the proline-rich segment and the SH3-like domain. The crystal structure of MntR (12) established structural homology between MntR and the N-terminal metal-and DNAbinding domain of DtxR (Figure 1). The residues at the C-terminus of MntR contribute to a dimer interface larger than that seen in DtxR and IdeR.…”

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confidence: 94%

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Mn(II) Binding by the Anthracis Repressor from Bacillus anthracis

et al. 2006

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The anthracis repressor (AntR) is a manganese-activated transcriptional regulator from Bacillus anthracis and is a member of the diphtheria toxin repressor (DtxR) family of proteins. In this paper, we characterize the Mn(II) binding and protein dimerization state using a combination of continuous wave (cw) and pulsed EPR methods. Equilibrium metal binding experiments showed that AntR binds 2 equivalents of Mn(II) with positive cooperativity and apparent dissociation constants of 210 and 16.6 µM. AntR showed sub-millisecond Mn(II) on-rates as measured using stopped-flow EPR. The kinetics of Mn(II) dissociation, measured by displacement with Zn(II), was biphasic with rate constants of 35.7 and 0.115 s -1 . Variable-temperature parallel and perpendicular mode cw EPR spectra showed no evidence of a spin-exchange interaction, suggesting that the two Mn(II) ions are not forming a binuclear cluster. Finally, size exclusion chromatography and double electron-electron resonance EPR demonstrated that AntR forms a dimer in the absence of Mn(II). These results provide insights into the metal activation of AntR and allow a comparison with related DtxR proteins.Bacteria, like all organisms, regulate the amount of transition metal ions in the cell. Of these metals, iron is particularly important as it is essential for viability, but accumulation of iron, especially in the ferrous [Fe(II)] form, results in oxidative damage as the metal is oxidized to the ferric form. Bacteria generally do not accumulate iron or other transition metals as seen in eukaryotic cells and, therefore, require efficient mechanisms for regulating the intracellular concentration of transition metal ions (1). Iron homeostasis is regulated in many bacteria by proteins of the diphtheria toxin repressor (DtxR) 1 family. These proteins function as metal ion sensors and regulate the transcription of genes involved in metal uptake, storage, and utilization.DtxR, the prototypical member of this family of repressors, regulates more than 40 genes in Corynebacterium diphtheriae, and IdeR, from Mycobacterium tuberculosis, regulates approximately 45 different genes (2). In many cases, these repressors have been co-opted to regulate the virulence response (1, 3). In C. diphtheriae, toxin gene expression is repressed by the metal-activated form of DtxR when the intracellular Fe(II) levels are above a certain threshold. A drop in the Fe(II) level inactivates the repressor and allows active gene transcription.DtxR proteins typically contain two domains connected by a flexible tether (Figure 1).

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Section: Discussionsupporting

confidence: 66%

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confidence: 94%

Mn(II) Binding by the Anthracis Repressor from Bacillus anthracis

et al. 2006

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“…MntR is a 142-amino acid protein that binds its DNA-recognition sequence as a homodimer in the presence of activating metal ions (12,13). Like other members of the DtxR family, MntR requires the binding of two metal ions per protein monomer and utilizes a helixturn-helix DNA-binding motif (12,13).…”

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

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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“…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.…”

Section: Introductionmentioning

confidence: 99%

“…2+ or Cd 2+ , another strongly activating metal ion. 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.…”

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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

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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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Structure of the manganese-bound manganese transport regulator of Bacillus subtilis

Cited by 92 publications

References 32 publications

Mn(II) Binding by the Anthracis Repressor from Bacillus anthracis

Mn(II) Binding by the Anthracis Repressor from Bacillus anthracis

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

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

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