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 ...

The streptococcal coaggregation regulator (ScaR) of Streptococcus gordonii is a manganese-dependent transcriptional regulator. When intracellular manganese concentrations become elevated, ScaR represses transcription of the scaCBA operon, which encodes a manganese uptake transporter. A member of the DtxR/MntR family of metalloregulators, ScaR shares sequence similarity with other family members, and many metal-binding residues are conserved. Here, we show that ScaR is an active dimer, with two dimers binding the 46-bp scaC operator. Each ScaR subunit binds two manganese ions, and the protein is activated by a variety of other metal ions, including Cd2+, Co2+ and Ni2+, but not Zn2+. The crystal structure of apo-ScaR reveals a tertiary and quaternary structure similar to its homolog, the iron-responsive regulator DtxR. While each DtxR subunit binds a metal ion in two sites, labeled primary and ancillary, crystal structures of ScaR determined in the presence of Cd2+ and Zn2+ show only a single occupied metal binding site that is novel to ScaR. The site analogous to the primary site in DtxR is unoccupied, and the ancillary site is absent from ScaR. Instead, metal ions bind to ScaR at a site labeled “secondary”, which is composed of Glu80, Cys123, His125 and Asp160 and lies roughly 5 Å away from where the ancillary site would be predicted to exist. This difference suggests that ScaR and its closely related homologs are activated by a mechanism distinct from that of either DtxR or MntR.

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MntR is a metalloregulatory protein that helps to modulate the level of manganese in Bacillus subtilis. MntR shows a metal-response profile distinct from other members of the DtxR family of metalloregulatory proteins, which are generally considered to be iron(II)-activated. As part of an ongoing effort to elucidate the mechanism and metal-selectivity of MntR, several biophysical studies on wild-type MntR and two active site mutants, MntR E99C and MntR D8M, have been performed. Using circular dichroism (CD) spectroscopy, the thermal stability of these proteins has been examined in the presence of various divalent metal ions. Fluorescence intensity measurements of 8-anilino-1-naphthalenesulfonic acid (ANS) were monitored to examine the folding of these proteins in the presence of different metal ions. These experiments indicate that MntR undergoes a significant conformational change upon metal binding that results in stabilization of the protein structure. These studies also show that the MntR D8M active site mutation causes a detrimental effect on the metal-responsiveness of this protein. Fluorescence anisotropy experiments have been performed to quantify the extent of metal-activated DNA binding by these proteins to two different cognate recognition sequences. Binding of MntR and MntR E99C to the mntA cognate sequence closely parallels that of the mntH operator, confirming that the proteins bind both sequences with comparable affinity depending on the activating metal ion. Fluorescence anisotropy experiments on MntR D8M indicate significantly impaired DNA binding, providing additional evidence that MntR D8M is a dysfunctional regulator.