Functional Studies of the Mycobacterium tuberculosis Iron-dependent Regulator

Chou, Chia Cheng; Wisedchaisri, Goragot; Monfeli, Ryan; Oram, Diana Marra; Holmes, Randall K.; Hól, Wim G. J.; Beeson, Craig C.

doi:10.1074/jbc.m407385200

Cited by 43 publications

(91 citation statements)

References 37 publications

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“…1) (16), again in a fashion similar to that of the Ms⌬ctpJ strain. These characteristics appear similar to those observed for PfteT, the single P 1B4 -ATPase present in B. subtilis (13) (47,48). Distinct from CtpJ, CtpD is not induced by divalent metals but by redox stressors, such as the nitric oxide generator nitroprusside and the respiratory poison cyanide (16).…”

Section: Discussionsupporting

confidence: 59%

Fine-tuning of Substrate Affinity Leads to Alternative Roles of Mycobacterium tuberculosis Fe2+-ATPases

Patel

Lewis

Long

et al. 2016

Journal of Biological Chemistry

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Little is known about iron efflux transporters within bacterial systems. Recently, the participation of Bacillus subtilis PfeT, a P 1B4 -ATPase, in cytoplasmic Fe 2؉ efflux has been proposed. We report here the distinct roles of mycobacterial P 1B4 -ATPases in the homeostasis of Co 2؉ Iron is an essential micronutrient required for numerous biological processes as it is used as a prosthetic group by several different enzymes (1, 2). However, in excess, it can be toxic due to its participation in Fenton chemistry and potential mismetallation in non-iron-containing metalloproteins. In this context, damage of iron-sulfur centers and mononuclear iron enzymes produced by various redox stresses are particular contributors to iron dyshomeostasis and consequent toxicity (3-6). Characterization of bacterial Fe 2ϩ homeostasis has mainly been focused in mechanisms of uptake (by divalent metal, siderophore, and heme transporters), transcriptional regulation (by Fur and IdeR systems), and Fe 2ϩ sequestration (by bacterioferritin and Dps proteins) (2, 7-9). Nevertheless, studies have suggested that cation diffusion facilitators and iron-citrate transporters participate in Fe 2ϩ efflux (10 -12). We recently observed that Bacillus subtilis PfeT, a P 1B4 -ATPase, confers Fe 2ϩ tolerance (13). PfeT is expressed under the control of PerR in response to peroxide exposure (14). Initial biochemical characterization showed that Fe 2ϩ activates isolated PfeT ATPase, leading to a higher V max than generated by Co 2ϩ , which is the proposed substrate of P 1B4 -ATPases (13,(15)(16)(17). Interestingly, phenotypic analysis of Listeria monocytogenes lacking the P 1B4 -ATPase FrvA showed a role of this ATPase in resistance to heme toxicity (18). These observations suggest a significant role of this subfamily of P-type ATPases in Fe 2ϩ homeostasis (13,14). P 1B4 -ATPases present in prokaryotes and plant chloroplasts are part of the large family of P-type ATPases (15,19,20). P-type ATPases are polytopic membrane proteins that transport a variety of ions using the energy provided by ATP hydrolysis (21-23). The P 1B subgroup includes proteins responsible for the efflux of cytoplasmic transition metals including Cu ϩ , Zn 2ϩ , Co 2ϩ , and Ni 2ϩ (19,22,23). The specificity of their transmembrane metal binding sites (TM-MBSs) 2 is determined by invariant amino acid sequences in their last three transmembrane segments (TMs) (17, 19, 24 -26). However, activation by non-cognate substrates has been reported for most P 1B -ATPase subgroups (22,27

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Section: Discussionsupporting

confidence: 59%

Fine-tuning of Substrate Affinity Leads to Alternative Roles of Mycobacterium tuberculosis Fe2+-ATPases

Patel

Lewis

Long

et al. 2016

Journal of Biological Chemistry

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“…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?…”

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

“…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).…”

mentioning

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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“…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%

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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Functional Studies of the Mycobacterium tuberculosis Iron-dependent Regulator

Cited by 43 publications

References 37 publications

Fine-tuning of Substrate Affinity Leads to Alternative Roles of Mycobacterium tuberculosis Fe2+-ATPases

Fine-tuning of Substrate Affinity Leads to Alternative Roles of Mycobacterium tuberculosis Fe2+-ATPases

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

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

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