Mn(II) Binding by the Anthracis Repressor from <i>Bacillus anthracis</i>

Sen, K. Ilker; Sienkiewicz, Andrzej; Love, J.; vanderSpek, Johanna C.; Fajer, Piotr G.; Logan, Timothy M.

doi:10.1021/bi052288g

Cited by 25 publications

(36 citation statements)

References 37 publications

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“…Two new reports describe some metal-binding affinities of MntR and a homolog AntR (15,18); our results are compared with these recent findings below. Measuring the metalbinding affinities is an essential component for understanding how MntR selectively responds to its cognate metal ion and elucidating its mechanism of action.…”

Section: Discussionmentioning

confidence: 52%

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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Section: Discussionmentioning

confidence: 52%

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

et al. 2006

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“…These observations are consistent with the previous solution studies showing that MntR retains dimeric structure in the presence and absence of activating metal ions. 3,5 In comparison to the fixed structure of the C-terminal domains in the MntR dimer, the positions of the N-terminal, DNA-binding domains vary greatly, via slight unwinding of the linker helix, α4, between residues 72-75 (Figure 2(a) and (b)). This behavior is similar to that observed among the multiple reported structures of the MntR•Mn 2+ complex, 18 though it is far more dramatic in the metal-free state.…”

Section: The Structure Of Apo-mntrmentioning

confidence: 99%

“…However, multiple lines of evidence suggest that the physiological complex places two Mn 2+ at a separation of 4.4 Å, bridged by Glu99 and Glu102. 5,17,18 Cadmium binds in a similar geometry to the two sites, which have been identified as A and C ( Figure 1). Interestingly, the structure of MntR complexed with Zn 2+ , a weakly activating metal, reveals only a single bound metal ion, in the A site.…”

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confidence: 93%

“…In several pathogenic species of bacteria, the regulation of manganese uptake has been linked to virulence. 4 Notably, Bacillus anthracis, the causative agent of anthrax, possesses a close homolog of MntR, 5 and mutants of B. anthracis lacking proteins involved in manganese uptake have weakened virulence. 6 Furthermore, MntR is a member of the DtxR/IdeR family of metalloregulators, named for the diphtheria toxin repressor (DtxR) of Corynebacterium diphtheriae and the iron-dependent regulator (IdeR) of Mycobacterium tuberculosis, both of which play key roles in the virulence of the organisms from which they have been isolated.…”

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confidence: 99%

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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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“…Spin-spin distances were measured by DEER with a Bruker E680-pulsed EPR spectrometer (Billerica, MA) at X-band (9.5 GHz) (55). One hundred-microliter samples were contained in 3-mm i.d.…”

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confidence: 99%

Spectroscopic validation of the pentameric structure of phospholamban

Traaseth¹,

Verardi

Torgersen

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

100

126

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Phospholamban (PLN) regulates calcium translocation within cardiac myocytes by shifting sarco(endo)plasmic reticulum Ca 2؉ -ATPase (SERCA) affinity for calcium. Although the monomeric form of PLN (6 kDa) is the principal inhibitory species, recent evidence suggests that the PLN pentamer (30 kDa) also is able to bind SERCA. To date, several membrane architectures of the pentamer have been proposed, with different topological orientations for the cytoplasmic domain: (i) extended from the bilayer normal by 50 -60°; (ii) continuous ␣-helix tilted 28°relative to the bilayer normal; (iii) pinwheel geometry, with the cytoplasmic helix perpendicular to the bilayer normal and in contact with the surface of the bilayer; and (iv) bellflower structure, in which the cytoplasmic domain helix makes Ϸ20°angle with respect to the membrane bilayer normal. Using a variety of cell membrane mimicking systems (i.e., lipid vesicles, oriented lipid bilayers, and detergent micelles) and a combination of multidimensional solution/solidstate NMR and EPR spectroscopies, we tested the different structural models. We conclude that the pinwheel topology is the predominant conformation of pentameric PLN, with the cytoplasmic domain interacting with the membrane surface. We propose that the interaction with the bilayer precedes SERCA binding and may mediate the interactions with other proteins such as protein kinase A and protein phosphatase 1.Ca 2ϩ -ATPase ͉ EPR ͉ membrane protein ͉ solid-state NMR ͉ protein dynamics C alcium translocation into the sarcoplasmic reticulum of cardiac myocytes is controlled by the sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA). Phospholamban (PLN) regulates the activity of SERCA by shifting the apparent calcium affinity for the enzyme. This activity is relieved by phosphorylation of PLN at Ser-16 and/or Thr-17 and high calcium concentration within the cytosol. Wild-type PLN (wt-PLN) forms stable homopentamers in lipid bilayers and in detergent micelles, where each monomer is composed of a helical cytoplasmic domain (residues 1-16), a semiflexible loop (residues 17-21), and a helical transmembrane domain (residues 22-52) (1, 2). Mutagenesis, molecular biology, and in vivo studies revealed that the PLN pentamer depolymerizes into active monomers that bind and inhibit SERCA (3). Similar conclusions were reached by in vitro fluorescence studies (4). Recently, Young and coworkers (5) have reported a cocrystal formed by SERCA and PLN pentamer, suggesting that the pentameric species also is able to bind SERCA. Furthermore, Jones and coworkers (6) hypothesized that the PLN pentamer may act as a chloride ion channel, which is supported by the bellflower structure recently determined by Oxenoid and Chou (2).There are four principal proposed structural models of pentameric wt-PLN, which differ primarily in the topology of the more dynamic cytoplasmic domain. In each of these models (shown in Fig. 1), residues 32-52 are in a coiled helix approximately parallel to the bilayer normal. The first model (extended helix/sheet ...

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

Cited by 25 publications

References 37 publications

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

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

Spectroscopic validation of the pentameric structure of phospholamban

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