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

Abstract

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...

Protein Dynamics and Monomer−Monomer Interactions in AntR Activation by Electron Paramagnetic Resonance and Double Electron−Electron Resonance

“…In a very recent study, a similar scenario for a member of the single domain family, MntR was proposed based on crystallographic results (45). DeWitt et al showed that MntR has a conformational distribution of the DNA binding sites in the apo form.…”

Physical Characterization of the Manganese-Sensing Pneumococcal Surface Antigen Repressor from Streptococcus pneumoniae

“…6A) against H–D exchange relative to the apo-state 59 and limits the conformational disorder of the DNA-binding domain relative to the dimerization domain. 24 In any case, what seems clear is that direct coordination by M8 in DtxR/IdeR and D7 in MntR by the activating metal drives a global “caliper-like” closure of the DNA-binding domains in each subunit relative to one another (Supplementary Fig. S1), which brings them into alignment to bind to successive major grooves in the DNA operator.…”

“…20,21 The three most extensively studied members of this family are the homologous Fe(II)-sensors Corynebacterium diphtheriae DtxR and Mycobacterium tuberculosis IdeR 22,23 and Mn(II)-sensor in Bacillus subtilis, MntR. 24–31 Although similar in both primary structure and quaternary structure of the activated metal-bound forms (see Supplementary Fig. S1), DtxR and MntR induce transcriptional co-repression by binding their cognate metal ions in distinct ways.…”

Opposite Effects of Mn2+and Zn2+on PsaR-Mediated Expression of the Virulence GenespcpA,prtA, andpsaBCAofStreptococcus pneumoniae

“…The conformation of Mn 2ϩ -bound MntR differs from the Zn 2ϩ -bound conformation with respect to the occupancy of the metal binding sites: Mn 2ϩ binds to two sites, whereas only one Zn 2ϩ ion binds to MntR, which does not allow binding of a second one (36). Metal binding at the second site is proposed to be required for DNA binding, as it promotes a disorder-to-order transition of MntR structure (17). PsaR shares 25% and 15% sequence identity with DtxR and MntR, respectively.…”