Roles of the A and C Sites in the Manganese-Specific Activation of MntR

McGuire, Amanda; Cuthbert, Bonnie J.; Ma, Zhen; Grauer-Gray, Kristen D.; Brophy, Megan Brunjes; Spear, Kayce A.; Soonsanga, Sumarin; Kliegman, J.I.; Griner, Sarah L.; Helmann, John D.; Glasfeld, Arthur

doi:10.1021/bi301550t

Cited by 39 publications

(63 citation statements)

References 52 publications

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“…The hexacoordinate geometry is achieved via additional ligating interactions from the backbone carbonyl of the residue equivalent to Glu99 and a solvent molecule that is missing in the SloR-Zn 2ϩ complex (10,34). In addition, a more distantly related DtxR family member, the Mn-specific MntR from B. subtilis, whose C site is composed of the same set of residues as the primary site of SloR, binds strongly activating metals with octahedral geometry (32,35). Mn 2ϩ typically prefers octahedral geometry in biological settings (36) and likely incorporates the backbone carbonyl of Glu99 as part of its coordination shell in the primary site of SloR.…”

Section: Discussionmentioning

confidence: 99%

Interactions of the Metalloregulatory Protein SloR from Streptococcus mutans with Its Metal Ion Effectors and DNA Binding Site

et al. 2015

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Streptococcus mutans is the causative agent of dental caries, a significant concern for human health, and therefore an attractive target for therapeutics development. Previous work in our laboratory has identified a homodimeric, manganese-dependent repressor protein, SloR, as an important regulator of cariogenesis and has used site-directed mutagenesis to map functions to specific regions of the protein. Here we extend those studies to better understand the structural interaction between SloR and its operator and its effector metal ions. The results of DNase I assays indicate that SloR protects a 42-bp region of DNA that overlaps the sloABC promoter on the S. mutans UA159 chromosome, while electrophoretic mobility shift and solution binding assays indicate that each of two SloR dimers binds to this region. Real-time semiquantitative reverse transcriptase PCR (real-time semiqRT-PCR) experiments were used to determine the individual base pairs that contribute to SloR-DNA binding specificity. Solution studies indicate that Mn 2؉ is better than Zn 2؉ at specifically activating SloR to bind DNA, and yet the 2.8-Å resolved crystal structure of SloR bound to Zn 2؉ provides insight into the means by which selective activation by Mn 2؉ may be achieved and into how SloR may form specific interactions with its operator. Taken together, these experimental observations are significant because they can inform rational drug design aimed at alleviating and/or preventing S. mutans-induced caries formation. IMPORTANCE This report focuses on investigating the SloR protein as a regulator of essential metal ion transport and virulence gene expression in the oral pathogen Streptococcus mutans and on revealing the details of SloR binding to its metal ion effectors and binding to DNA that together facilitate this expression. We used molecular and biochemical approaches to characterize the interaction of SloR with Mn2؉ and with its SloR recognition element to gain a clearer picture of the regulatory networks that optimize SloRmediated metal ion homeostasis and virulence gene expression in S. mutans. These experiments can have a significant impact on caries treatment and/or prevention by revealing the S. mutans SloR-DNA binding interface as an appropriate target for the development of novel therapeutic interventions. Cariogenesis is a disease that derives from interactions involving the human dentition, an acidogenic microflora, including Streptococcus mutans, and dietary factors. Specifically, S. mutans, which is among the most cariogenic of the oral microbes, assumes an obligate biofilm lifestyle upon colonizing the tooth surface during the first year of life, shortly after tooth eruption. Commonly found in the human oral cavity as the most abundant species in the dental plaque biofilm, S. mutans metabolizes exogenous dietary carbohydrates to generate energy for itself via fermentation and releases acid as a metabolic byproduct. The buffering capacity of saliva combined with appropriate oral hygiene practices can maintain the p...

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Section: Discussionmentioning

confidence: 99%

Interactions of the Metalloregulatory Protein SloR from Streptococcus mutans with Its Metal Ion Effectors and DNA Binding Site

et al. 2015

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“…Although various Mn 2ϩ binding affinities have been measured for MntR, recent studies reveal an overall K d of ϳ6 M (4). Cd 2ϩ can also bind to MntR to repress the MntR regulon (49,57). Although Cd 2ϩ is considered a non-cognate metal with respect to Mn 2ϩ homeostasis, Cd 2ϩ may be a physiologically relevant agonist; MntH is a major route of entry for the toxic cadmium ion (11).…”

Section: Selective Recognition Of Manganese By Mntrmentioning

confidence: 99%

“…Numerous structures are available for B. subtilis MntR as an apo-protein (56), in various metallated states (51,55,57), and for mutant proteins altered in metal recognition (57). Collectively, these structures lead to a model for manganese-selective metallosensing in which manganese binds first to the A site with heptacoordinate ligation, and this organizes the C site to enable binding of a second manganese and stabilization of the active repressor conformation (Fig.…”

Section: Selective Recognition Of Manganese By Mntrmentioning

confidence: 99%

“…MntR also binds tightly to non-cognate metals (Fe 2ϩ , Co 2ϩ , and Zn 2ϩ ), but these fail to allosterically activate DNA binding due to an inability to form a binuclear cluster (57). Fe 2ϩ binds at the A site but with a different geometry than manganese, and this distorts the C site such that the second metal binding event (required for DNA binding) is inhibited (57). As a result, Fe 2ϩ is an antagonist for MntR (11,52,54).…”

Section: Selective Recognition Of Manganese By Mntrmentioning

confidence: 99%

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Specificity of Metal Sensing: Iron and Manganese Homeostasis in Bacillus subtilis

Helmann

2014

Journal of Biological Chemistry

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Metalloregulatory proteins allow cells to sense metal ions and appropriately adjust the expression of metal uptake, storage, and efflux pathways. Bacillus subtilis provides a model for the coordinate regulation of iron and manganese homeostasis that involves three key regulators: Fur senses iron sufficiency, MntR senses manganese sufficiency, and PerR senses the intracellular Fe/Mn ratio. Here, I review the structural and physiological bases of selective metal perception, the effects of non-cognate metals, and mechanisms that may serve to coordinate iron and manganese homeostasis. Manganese and Iron: Chemically Similar Elements with Distinct Roles in Cell PhysiologyManganese and iron are both predominantly present as divalent cations in the reducing environment of the cytoplasm and are maintained at overall concentrations (averaged over the cell) that can approach 0.5 mM (1-3). Much of this manganese and iron is bound by enzymes that acquire metal from a kinetically accessible (labile) pool buffered in the low M range (4 -6). These two metals are not only important from the perspective of individual microbial cells, but have a global impact due to their central roles in photosynthesis, nitrogen fixation, and other key steps in elemental cycling.Nearly all cellular life requires significant amounts of iron. The sole documented exceptions are the lactobacilli and the spirochete Borrelia burgdorferi, which have little if any requirement for iron (3). Coincident with this disregard for iron, these same organisms have high requirements for manganese and have been described as manganese-centric (7). Conversely, other organisms have little demonstrable requirement for manganese, although they may use manganese when available. The best characterized is Escherichia coli, which has a largely ironcentric metabolism, but conditionally imports manganese in response to oxidative stress (8).Here, I review the key factors regulating iron and Mn homeostasis in the model organism Bacillus subtilis. B. subtilis encodes three members of the Fur 2 family (Fur, PerR, and Zur) and one member of the DtxR/MntR family (MntR) that regulate the import of nutrient metal ions (9 -11). Structural and biochemical studies have provided insights into the mechanisms of selective activation of these repressors by their cognate metal ions (12-14). B. subtilis requires both iron and manganese for growth, a property shared with many pathogens for which the ability to obtain these metals from the host is a critical determinant of virulence (7,(15)(16)(17). Metalloregulators as a Window into Cellular PhysiologyMetalloregulators function as the arbiters of metal ion sufficiency and excess (6,12,18). For those that sense sufficiency, the affinity (measured as K d ) for their cognate metal(s) defines the level of metal judged to be sufficient. When free metal concentrations rise above this level, the regulator transitions from its inactive (apo-) form to its activated (holo-) form and represses expression of metal import. In B. subtilis, Fur serves as ...

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“…The most recent studies on DtxR and its homologues have delved into the structural specifics of these proteins, often using crystal structures to explore mechanisms of metal ion selectivity, metal ion binding, and DNA consensus sequence binding. As a wealth of literature has been dedicated to these matters (see Glasfield et al, 2003 Haswell et al, 2013;McGuire et al, 2013), this review instead approaches metalloregulation from a pathogenesis perspective, with an evolutionary and ecological emphasis. While recognizing that metalloregulation represents just one dimension of virulence gene control, we nonetheless collate a number of studies on DtxR, SloR, MtsR, and MntR to draw broad conclusions about the role of metalloregulation in bacterial pathogenesis.…”

Section: Introductionmentioning

confidence: 99%

A role for the DtxR family of metalloregulators in gram-positive pathogenesis

Merchant

Spatafora

2013

Mol oral Microbiol

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SUMMARYGiven the central role of transition metal ions in a variety of biochemical processes, the colonization, survival, and proliferation of a bacterium within a host hinges upon its ability to overcome the metal ion deprivation that characterizes nutritional immunity. Metalloregulatory, or "metal-sensing" proteins have evolved in bacteria to mediate metal ion homeostasis by activating or repressing the expression of genes encoding metal ion transport systems upon binding their cognate metal ion. Yet increasing evidence in the literature supports an additional role for these metalloregulatory proteins in pathogenesis. Herein, we survey studies on the DtxR family of metalloregulators, namely DtxR (Cornyebacterium diphtheriae), SloR (Streptococcus mutans), MtsR (Streptococcus pyogenes), and MntR (Staphylococcus aureus) to describe how metalloregulation enables adaptive virulence gene expression within the mammalian host. This research has important implications for drug design, as the generation of hyper-repressive metalloregulatory proteins may represent a mechanism by which to attenuate bacterial pathogenicity. The fact that metalloregulators are unique to prokaryotes makes these proteins especially attractive therapeutic targets.

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Roles of the A and C Sites in the Manganese-Specific Activation of MntR

Cited by 39 publications

References 52 publications

Interactions of the Metalloregulatory Protein SloR from Streptococcus mutans with Its Metal Ion Effectors and DNA Binding Site

Interactions of the Metalloregulatory Protein SloR from Streptococcus mutans with Its Metal Ion Effectors and DNA Binding Site

Specificity of Metal Sensing: Iron and Manganese Homeostasis in Bacillus subtilis

A role for the DtxR family of metalloregulators in gram-positive pathogenesis

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