Structural analysis of the manganese transport regulator MntR from Bacillus halodurans in apo and manganese bound forms.

The manganese transport regulator MntR is a metal-ion activated transcriptional repressor of manganese transporter genes to maintain manganese ion homeostasis. MntR, a member of the diphtheria toxin repressor (DtxR) family of metalloregulators, selectively responds to Mn2+ and Cd2+ over Fe2+, Co2+ and Zn2+. The DtxR/MntR family members are well conserved transcriptional repressors that regulate the expression of metal ion uptake genes by sensing the metal ion concentration. MntR functions as a homo-dimer with one metal ion binding site per subunit. Each MntR subunit contains two domains: an N-terminal DNA binding domain, and a C-terminal dimerization domain. However, it lacks the C-terminal SH3-like domain of DtxR/IdeR. The metal ion binding site of MntR is located at the interface of the two domains, whereas the DtxR/IdeR subunit contains two metal ion binding sites, the primary and ancillary sites, separated by 9 Å. In this paper, we reported the crystal structures of the apo and Mn2+-bound forms of MntR from Bacillus halodurans, and analyze the structural basis of the metal ion binding site. The crystal structure of the Mn2+-bound form is almost identical to the apo form of MntR. In the Mn2+-bound structure, one subunit contains a binuclear cluster of manganese ions, the A and C sites, but the other subunit forms a mononuclear complex. Structural data about MntR from B. halodurans supports the previous hypothesizes about manganese-specific activation mechanism of MntR homologues.

Introduction

Metal ions are essential for living organisms because iron, zinc, and n class="Chemical">manganese ions act as cofactors for many proteins which are involved in photosynthesis, nerve transmission, and defense against toxins[1]. Manganese ions are important in many fundamental cellular processes, including protection against oxidative stress and the synthesis of the deoxyribonucleotides required for DNA replication[2,3]. However, and excess of manganese ions can be toxic[4,5]. Therefore, in order to maintain homeostasis, it is important for cells to sense and respond to manganese ion concentrations[6,7]. Metalloregulatory proteins regulate metal ion homeostasis in bacteria by binding metal ions, leading to the activation or repression of the transcription of genes involved in import or efflux of the ions[8,9]. Each metalloregulatory protein has a different ligand selectivity for allosteric activation[10].

The transcriptional regulation and manganese binding of MntR from n class="Species">Bacillus subtilis has been well studied. The manganese transport regulator (MntR) functions as a homodimer and is activated by Mn2+ to repress the expression of two manganese uptake systems, MntABCD and MntH, in response to elevated concentrations of Mn2+[11]. Recent studies have shown that MntR activates the expression of two efflux systems, MneP and MneS, in Bacillus subtilis[9]. MntR is a member of the DtxR/IdeR family, which maintains iron ion homeostasis in bacteria[12]. Corynebacterium diphtheriae DtxR and Mycobacterium tuberculosis IdeRs consist of three domains: an N-terminal HTH-motif DNA binding domain (domain 1), a dimerization domain (domain 2), and a C-terminal SH3-like domain (domain 3), which is absent in MntR family proteins[13-15]. MntR consists of two domains: an N-terminal HTH-motif DNA binding domain (domain 1) and a C-terminal dimerization domain (domain 2)[5]. The DtxR/IdeR family proteins have two major metal binding sites 9.0 Å apart, called the primary and ancillary sites[7,16]. MntR is shorter than DtxR/IdeR family and the ancillary site of MntR is absent, because of the lack of an SH3-like domain in MntR[8]. The metal binding site of MntR is located between domains 1 and 2, corresponding to the primary site in DtxR/IdeR[7]. From previous structural studies it is known that the metal binding site of B. subtilis MntR consists of several residues including Asp8 and Glu11 in domain 1, and His77, Glu99, Glu102 and His103 in domain 2. There are two types of metal ion binding conformations in MntR, the AB conformer, and the AC conformer, resulting from differences in amino acid residues involved in metal coordination and distances between the two metal ions[5]. In the AB conformer, Asp8, Glu11, Glu102 and His103 interact with a B site Mn2+ ion, and the metal binding sites are separated 3.3 Å. In contrast, Asp8, Glu99, Glu102 and His103 interact with a C site Mn2+ ion, and the sites are separated 4.4 Å in the AC conformer[4].

The metal coordination geometry of MntR is essential for the generation of selective responses to cognate metals. Larger metal cations (Mn2+ and n class="Chemical">Cd2+) form a binuclear complex with MntR and are fully activated. However, when bound to small metal cations (Fe2+, Co2+, and Zn2+), the metal ions do not fully occupy the site, but form a mononuclear complex, resulting in low activity[17].

The crystal structures of the MntR family have been determined from several bacterial species, including Bacillus subtilis[18], n class="Species">Escherichia coli[7], and Mycobacterium tuberculosis[17]. Previous structural studies of the MntR family have described how conformation changes depending on whether the sites are bound to cognate metal ions, and how such conformational changes induce a dissociation of cognate DNA from the MntR protein[19]. The MntR homologue (BH2807, BhMntR) in Bacillus halodurans is a protein consisting of 139 amino acids, and has 78% sequence identity with MntR from B.subtilis (BsMntR). Further sequence comparisons of B. halodurans MntR show that it is 31% identical to E. coli MntR, 26% identical to M. tuberculosis MntR, 24% identical to C. diphtheriae DtxR, and 26% identical to T. acidophilum IdeR (Fig 1A).

Fig 1

Multiple sequence alignment and overall structure of BhMntR.

(A) Multiple sequence alignment of BhMntR with other MntR homologues. The secondary structures of BhMntR are indicated above the sequence. The highly conserved and partially conserved residues are shaded in black and gray boxes, respectively. The residues involved in metal binding are shown as red triangles at the bottom of the sequence. (B) The monomeric and dimeric structures of apo BhMntR. BhMntR are composed of an N-terminal DNA binding domain (yellow) and a C-terminal domain (green). (C) The dimeric structure of Mn2+-bound form BhMntR. One subunit contained binuclear manganese ions (purple), while the other subunit forms a mononuclear complex with magnesium ion (gray).

(A) Multiple sequence alignment of BhMntR with other MntR homologues. The secondary structures of BhMntR are indicated above the sequence. The highly conserved and partially conn class="Chemical">served residues are shaded in black and gray boxes, respectively. The residues involved in metal binding are shown as red triangles at the bottom of the sequence. (B) The monomeric and dimeric structures of apo BhMntR. BhMntR are composed of an N-terminal DNA binding domain (yellow) and a C-terminal domain (green). (C) The dimeric structure of Mn2+-bound form BhMntR. One subunit contained binuclear manganese ions (purple), while the other subunit forms a mononuclear complex with magnesium ion (gray).

Although the crystal structures of MntR from bacterial species have been determined, the metal coordination and selectivity are not fully understood. To further understand the metal binding site of the MntR protein, we determined crystal structures of the apo and manganese-bound forms of MntR from n class="Species">B. halodurans. The structures revealed that BhMntR forms a binuclear complex with manganese ions in the AC conformer.

Materials and methods

Expression and purification of BhMntR

The mntR genes were amplified using polymerase chain reaction (PCR) using the genomic DNA of n class="Species">B. halodurans as a template. The amplified mntR genes were inserted into an NdeІ/XhoІ-digested vector pET-28b(+) (Novagen, Germany) producing a hexahistidine-tag (His-tag) at its N-terminus. The recombinant BhMntR was transformed and expressed in E.coli BL21(DE3) Star pLysS cells (Invitrogen, USA). The transformed cells were grown at 310 K to an OD600 of ~0.5 in Luria-Bertani medium supplemented with 30 μg mL-1 kanamycin and chloramphenicol. Overexpression of recombinant BhMntR was induced with 1.0 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and allowed to grow for four hours at 303 K. The cells were harvested by centrifugation at 4,200 g for 15 minutes at 277 K and frozen immediately at 193 K. The cell pellets (6~8 g) were resuspended in buffer A (20 mM Tris-HCl pH 8.0, 0.5 M NaCl, and 10%(v/v) glycerol) containing 1 mM phenylmethylsulfonyl fluoride and homogenized using an ultrasonic processor (Sonics & Materials™, Vibra Cell VCX 750, USA). The insoluble fraction was removed by centrifugation at 28,000 g (Supra 22 K; Hanil BioMed Inc., Korea) for one hour at 277 K.

The recombinant BhMntR in the soluble fraction was loaded on a nickel-charged His-trap immobilized metal affinity chromatography (IMAC) column (GE Healthcare, UK) pre-equilibrated with buffer A, washed with buffer A containing 60 mM n class="Chemical">imidazole, and eluted from the column with buffer B (20 mM Tris-HCl pH 8.0, 0.5 M NaCl, 10%(v/v) glycerol, and 300 mM imidazole) to immobilized-metal-affinity-chromatography (IMAC) on a Ni-NTA resin (GE Healthcare). The BhMntR was further purified by size exclusion chromatography using a Superdex 200 gel-filtration column (GE Healthcare, UK), employing with elution buffer (20 mM Tris-HCl pH 8.0, 0.2 M NaCl, 5% (v/v) glycerol, 1 mM dithiothreitol (DTT), and 2 mM MgCl2). The purity of BhMntR was assessed using 12% (v/v) SDS-PAGE. The purified BhMntR was concentrated to 17 mg/ml using centrifugal filter units (Millipore) and aliquots of the protein were stored at 193 K.

Crystallization and X-ray diffraction data collection

Crystallization of BhMntR was performed using the sitting-drop vapor diffusion method at 296 K with 96-well crystallization plates (SWISSCI n class="CellLine">MRC, UK) and commercial screening solution from Anatrace, Hampton Research, Emerald Biosystems and Molecular Dimensions. Each sitting-drop was prepared by mixing 0.75 μl of the concentrated protein and the reservoir solution. The crystals of apo BhMntR were grown in reservoir solution containing 0.1 M sodium/potassium phosphate pH 6.2 and 0.4 M magnesium formate. The co-crystallization of BhMntR with manganese ions was unsuccessful. Mn2+-bound crystals were obtained by soaking with 50 mM MnCl2 for one hour in apo crystals, grown in 0.1 M sodium phosphate pH 6.5 and 0.4 M magnesium formate.

Each crystal was transferred into a cryo-protectant solution containing the reservoir solution with 20%(v/v) n class="Chemical">glycerol and flash-cooled in a liquid nitrogen stream. X-ray diffraction data were collected at 100 K with a Pilatus3 6M detector using synchrotron radiation on a Beamline 11C of the Pohang Accelerator Laboratory (PAL) in Korea. The crystals were exposed to X-rays for 1.0 second per image, and 180 frames were obtained for each 1.0° oscillation. All data were processed and scaled using DENZO and SCALEPACK from the HKL-2000 program suite[20]. The detailed data collection statistics are summarized in Table 1.

Table 1

Data collection and refinement statistics.

	Apo BhMntR	Mn2+-bound BhMntR
Data collection
Space group	P212121	P212121
Unit-cell parameters
a, b, c (Å)	39.66, 89.20, 109.76	39.53, 89.37, 109.95
α, β, γ (°)	90.00, 90.00, 90.00	90.00, 90.00, 90.00
Wavelength (Å)	0.97941	0.97960
Resolution (Å)	50.00–2.30 (2.34–2.30)	50.00–2.50 (2.54–2.50)
Number of observations	115,264	93,537
Unique reflections	17,942	14,131
Data completeness (%)	99.1 (99.2)	99.9 (99.9)
Redundancy	6.4 (6.0)	6.6 (7.1)
Averge I/σ(I)	16.2 (4.5)	14.1 (7.9)
Rmerge (%)a	11.9 (36.4)	12.8 (36.7)
Refinement statistics
Resolution (Å)	46.74–2.30	37.20–2.50
Rwork / Rfree (%)	18.9/22.9	17.1/21.8
No. of non-H atoms	2439	2379
Protein	2280	2284
Ligands	24	18
Water	135	77
rmsd bonds (Å)	0.008	0.008
rmsd angles (°)	0.938	0.930
Average B-factor	35.7	27.9
Protein	28.48	21.65
Ligands	49.57 (glycerol)	47.20 (Mn2+,Mg2+,Phosphate) MGGMGD
Water	38.97	30.07
Ramachandran plot (%)
Favored	98.53	98.53
Allowed	1.47	1.47
Outliers	0	0

aRmerge = ΣhΣi|I(h)i − < I(h) > |/ΣhΣiI(h)i, where I(h) is the intensity of reflection h, Σh is the sum over all reflections, and Σi is the sum over i measurements of reflection h.

Structure determination and refinement

The structure of apo BhMntR was solved by molecular replacement using the program PHASER MR from the CCP4 program suite[21] using the apo BsMntR structure (PDB code 2HYG)[18] as a search model. The initial model was further improved by alternating cycles of manual building using the COOT program[22], and the model was refined with the n class="Chemical">PHENIX program package[21]. The refined model was evaluated using MolProbity[23]. The refinement statistics of apo BhMntR and Mn2+ bound BhMntR are presented in Table 1.

Results and discussion

Model building and quality

The apo crystal structure of BhMntR was determined at 2.3 Å resolution using molecular replacement with the MntR model of n class="Species">B. subtilis (2HYG). The structure was refined to crystallographic Rwork and Rfree values of 18.9% and 22.9%, respectively with good geometry. The refined model (PDB code 6KTA) contained two BhMntR subunits which formed a homodimer, four molecules of glycerol, and 135 water molecules in the asymmetric unit. The model was validated using MolProbity[23]. The C-terminal region of chains A (residue 139) and B (residues 136–139) were poorly ordered, due to lack of electron-density maps. Mn2+-bound BhMntR crystals were obtained by soaking with 50 mM MnCl2 for one hour in apo crystals. The crystal structure of the Mn2+-bound form was determined at 2.5 Å resolution, and the binuclear manganese ions were clearly evident in the 2Fo-Fc map and omit maps, whereas a magnesium ion was observed in the other subunit. The structure of Mn2+-bound BhMntR was refined with a crystallographic Rwork value of 17.1% and an Rfree value of 21.8%. Each subunit of the Mn2+-bound BhMntR was well defined, except for the C-terminal residue 139. The refined model (PDB code 6KTB) contains two BhMntR subunits, three molecules of phosphate, and 77 water molecules in the asymmetric unit. All refined models for BhMntR showed favored or allowed regions in a Ramachandran plot.

Overall structure of B. halodurans MntR

Each BhMntR subunit was composed of seven α-helices and two β-strands, which could be divided into an N-terminal Helix-Turn-Helix (HTH) DNA binding domain (domain1, residues 1–71) and a C-terminal dimerization domain (domain2, residues 72–139) (Fig 1). The N and C-terminal domains were connected by a long linker helix (α4) that extended from the wing to the dimer interface. The BhMntR was a homodimeric structure, with approximate dimensions of 40Å × 55Å × 80Å. The N-terminal DNA binding domain consisted of three α-helices and two strands of antiparallel β-sheet, forming a winged HTH motif that putatively interacted with DNA. Because helix α3 of the HTH motif could be responsible for DNA recognition, we speculate that the positively charged residues (Lys41, Lys45, and Lys48) in helix α3 are involved in DNA binding. Domain 2, the dimerization domain, is composed of four α-helices (α4–α7). Domains 1 and 2 are connected by the long linker helix α4 (residues 64–87).

The two subunits form a dimeric structure, related by a non-crystallographic 2-fold axis (Fig 1). The buried surface area of the dimer is about 1300 Å2, approximately 14% of the monomer surface area. The dimeric BhMntR is stabilized by the hydrogen bonds and hydrophobic interactions along helices α4 to α7; 14 residues were involved in hydrophobic interactions and eight residues in n class="Chemical">hydrogen bonds. (PDBePISA protein–protein interaction server: http://www.ebi.ac.uk/msd-srv/prot_int/ and PDBsum generate: http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.html). The dimer interface is mainly produced by hydrophobic side chains such as Phe83, Ile87, Gly88, Val 89, Gly100, Ile101, Leu105, Ala109, Ile113, Leu116, Tyr119, Phe120, Leu130, and Val133. Ten hydrogen bonds were formed between Asp90 N and Asp108 Oδ2, between Asp97 Oδ1 and Ser106 N, between Asp97 Oδ2 and Ser106 Oγ, between Tyr119 Oη and Tyr119 Oη, between Glu122 Oε1 and Lys136 Nζ, between Asp115 Oδ1 and Asn137 Nδ2, and between Gln118 Oε1 and Asn137 Nδ2. This finding demonstrated that BhMntR exists as a functional dimer in solution. Two subunits in the asymmetric unit of BhMntR showed little structural difference, with a root-mean-sequare deviation (r.m.s.d.) value of 1.31 Å for 137 Cα atoms in residues 1–137 (S1 Fig). There were few structural differences between apo and Mn2+-bound dimeric forms, with a r.m.s.d. value of 0.49 Å for 276 Cα atoms.

Metal binding site

We obtained Mn2+-bound crystals by soaking with 50 mM n class="Chemical">MnCl2 in apo crystals, and confirmed using an omit map and an anomalous map showing two peaks at the counter levels even at 5σ (Fig 2A and S2 Fig). The metal binding site appeared to be fully occupied in one subunit with the temperature factors for the two manganese ions being 51.02 Å2 and 63.88 Å2, respectively. However, the other subunit contained a magnesium ion which was coordinated by the side chains of Glu99, Glu102, and two water molecules (Fig 2C). The two manganese ions were found at the interface between the HTH domain and the dimerization domain and formed a binuclear complex separated by 4.5 Å, labeled as the A and C sites (AC conformer).

Fig 2

Metal ion binding site in the B. halodurans MntR.

(A) Stereoview of metal binding site in the B. halodurans MntR. A σA-weighted electron density map (2Fo-Fc map) contoured at 1.0σ (blue). Omit map was calculated, contoured at 3σ (red). The Mn2+ atoms (purple) are depicted with surrounding residues (yellow sticks from domain1 and green sticks from domain2). (B) Metal binding site with binuclear manganese ions. The coordination with binuclear manganese ions and the distance between Mnc and the backbone carbonyl oxygen of Glu99 are shown in yellow and red, respectively. (C) Metal binding site with a magnesium ion (gray). Unlike binuclear manganese ions binding site, a magnesium ion was coordinated by the side chains of Glu99, Glu102, and two water molecules. The His77 made a hydrogen bond with Glu81 via a water. The symmetry-related Tyr57 is colored gray.

(A) Stereoview of metal binding site in the B. halodurans MntR. A σA-weighted electron density map (2Fo-Fc map) contoured n class="Species">at 1.0σ (blue). Omit map was calculated, contoured at 3σ (red). The Mn2+ atoms (purple) are depicted with surrounding residues (yellow sticks from domain1 and green sticks from domain2). (B) Metal binding site with binuclear manganese ions. The coordination with binuclear manganese ions and the distance between Mnc and the backbone carbonyl oxygen of Glu99 are shown in yellow and red, respectively. (C) Metal binding site with a magnesium ion (gray). Unlike binuclear manganese ions binding site, a magnesium ion was coordinated by the side chains of Glu99, Glu102, and two water molecules. The His77 made a hydrogen bond with Glu81 via a water. The symmetry-related Tyr57 is colored gray.

The binuclear manganese ions were liganded by six amino acid residues: Asp8 and Glu11 contributed by domain 1, and His 77, n class="Chemical">Glu99, Glu102, and His103 contributed by domain 2 (Fig 2B). The two manganese ions (MnA and MnC) were jointly coordinated by the carboxylate oxygens of Glu99 and Glu102 from domain 2. Each metal ion was individually coordinated by Glu11 (MnA), His77 (MnA), His103 (MnC) and Asp8 (MnC). The MnA ion was coordinated by seven atoms: Glu11 Oε1/Oε2, His77 Nδ1, Glu99 Oε2, Glu102 Oε1/Oε2, and Wat95 O. In addition, the His77 Nε2 made a hydrogen bond with Glu81 Oε1, while the His77 Nδ1 in the other subunit made a hydrogen bond with Glu81 Oε1 via a water molecule (Fig 2C). The Mnc ion was coordinated by five atoms: Asp8 Oδ1, Glu99 Oε1, Glu102 Oε2, His103 Nε2, and Wat37 O, while the C site of BsMntR has octahedral coordination geometry. In the Mn2+-bound BsMntR structure, the backbone carbonyl oxygen of Glu99 coordinated with the Mnc ion, but this interaction between them was too distant to interact in the BhMntR, at 3.5 Å (Fig 2B).

In the other subunit of the Mn2+-bound MntR structure, no n class="Chemical">manganese binding was observed, although the residues are positioned appropriately to form a manganese binding site. The reason for the lack of bound manganese ions at this site is unclear. The side chain of His77, which is strictly conserved in the MntR/IdeR family, had a different rotamer with a hydrogen bond via a water molecule to Glu81 and was also stabilized by π-π interaction with symmetry-related Tyr57 (Fig 2C). These interactions could block the proper rotamer of His77 to coordinate with MnA ion in this subunit. These findings suggested that the His77 flip in BhMntR could initiate metal binding in the presence of manganese ions. It will be valuable to verify the role of His77 at the metal binding site in the future experiments.

Structural comparison to other MntR homologue

We carried out structural and sequence comparisons among DtxR/MntR proteins from various organisms using the Clustal Omega[24] and DALI server[25]. The best five matches were those of the metal-dependent DtxR/MntR family. They were (1) the n class="Chemical">manganese transport regulator, MntR from B. subtilis[4] (PDB code 2F5F; r.m.s. d. of 1.2 Å for 137 equivalent Cα positions in residue 2–138 of BhMntR, a Z-score of 19.1, and a sequence identity of 78%), (2) the MntR from E. coli[7] (PDB code 2H09; r.m.s.d. of 2.1 Å for 118 equivalent Cα positions in residue 1–114 and 116–119 of BhMntR, a Z-score of 15.7, and a sequence identity of 35%), (3) the C. diphtheriae DtxR in complex with DNA[26] (PDB code 1BI2; r.m.s. d. of 2.2 Å for 119 equivalent Cα positions in residue 1–119 of BhMntR, a Z-score of 14.1, and a sequence identity of 26%), (4) the M. tuberculosis IdeR in complex with DNA[27] (PDB code 1U8R; r.m.s.d. of 1.8 Å for 116 equivalent Cα positions in residue 3–119 of BhMntR, a Z-score of 13.8, and a sequence identity of 28%), and (5) the T. acidophilum IdeR in complex with DNA[28] (PDB code 4O6J; r.m.s.d. of 2.5 Å for 114 equivalent Cα positions in residue 4–118 of BhMntR, a Z-score of 12.7, and a sequence identity of 29%).

Previous studies revealed that BsMntR shows conformational changes when bound to the manganese ions by inducing a hinge bending motion between residues 72 and 75[18]. To investigate the hinge motion properties of BhMntR, we compared the domain orientation, by superimposing the Cα atoms of domain 2 (72–139) in the BhMntR structure with those of the apo BsMntR (PDB code 2HYG), the n class="Chemical">Mn2+-bound BsMntR (PDB code 2F5D), and the Zn2+-bound BsMntR (PDB code 2EV6). The r.m.s deviations in Cα positions for domain 2 (residues 72–139) are 0.90 Å, 0.76 Å and 0.95 Å (S1 Table). When the dimerization domain is superimposed, the DNA binding domains varies by 2.4–8.5 Å at residue Lys41. The movement of the DNA binding domain with respect to domain 2 is centered at residue Tyr75 of helix α4, and is tilted by 4.5–17° (Fig 3A). There is no loss of hydrogen bonding within helix α4 upon metal binding, while hydrogen bonding was lost within helix α4 in T. acidophilum IdeR. When measured between the Cα atoms of Lys41, at the center of helix α3, the domain separation of apo and manganese bound BhMntR are 37.4 Å and 37.5 Å, respectively, while the distance between the Lys41 in apo, Mn2+-bound, and Zn2+-bound BsMntR are 39.2 Å, 32.1Å and 30.7Å, respectively (Fig 3B). There was little domain movement between apo and Mn2+-bound BhMntR, possibly due to crystal packing or the presence of positively charged ions of Na+ (~0.5 M) and Mg2+ (~0.4 M) during the crystallization process. It will be important to verify the domain movement upon metal binding by co-cystallization in future experiments.

Fig 3

Structural comparison between BhMntR and BsMntR.

(A) Superimposing apo BhMntR with Mn2+-bound BhMntR and BsMntR. Superimposition was based on the dimerization domain of one subunit. The angle was centered at residue Tyr75 of the helix α4 and measured between the residues of Lys65. The apo BhMntR, Mn2+-bound BhMntR, apo BsMntR and Mn2+-bound BsMntR are indicated in green, yellow, cyan and magenta, respectively. (B) Distances of residue Lys41 in the dimeric structure. The dimeric structures of MntR are aligned by the dimerization domain.

(A) Superimposing apo BhMntR with Mn2+-bound BhMntR and BsMntR. Superimposition was based on the dimerization domain of one subunit. The angle was centered at residue Tyr75 of the helix α4 and measured between the residues of Lys65. The apo BhMntR, n class="Chemical">Mn2+-bound BhMntR, apo BsMntR and Mn2+-bound BsMntR are indicated in green, yellow, cyan and magenta, respectively. (B) Distances of residue Lys41 in the dimeric structure. The dimeric structures of MntR are aligned by the dimerization domain.

Conclusions

We reported the crystal structures of BhMntR: apo, and Mn2+-bound forms. Our results showed that BhMntR is composed of two distinct domains in the homodimeric form, and its overall structure is similar to those of other MntR homologues. The two n class="Chemical">manganese ions formed a binuclear cluster in the metal binding site of BhMntR, via six amino acid residues; three strictly conserved residues (His77, Glu102 and His103) in the IdeR/MntR family, two residues (Asp8 and Glu99) conserved in the MntR family, and a Glu11 conserved in MntR from B. subtilis and E. coli. The manganese ion in A site was liganded with heptageometry as shown in BsMntR, whereas the manganese ion in the C site was incompletely liganded with five atoms. The sixth atom, the carbonyl oxygen of Glu102, was too far away to coordinate with the MnC ion. Therefore, BhMntR did not cause movement of the domain to bind DNA upon manganese ion binding. Binuclear metal ions were not formed in the other subunit due to the crystal packing and the flipping of His77. The side chain of His77 was flipped and stabilized by hydrogen bonding and hydrophobic stacking. In order to initiate metal binding, the side chain of His77 was flipped to interact with the carboxylate of Glu81. Although the functional assignment of metal binding site for BhMntR is tentative, this structural model is applicable to other MntR homologous structures.

R.m.s.d plot of BhMntR.

(TIF)

Click here for additional data file.

Anomalous maps in metal ion binding site of BhMntR.

(A) Stereoview of metal binding site with binuclear manganese ions of the n class="Chemical">Mn2+-bound BhMntR. A σA-weighted electron density map (2Fo-Fc map) contoured at 1.0σ (blue). Anomalous map was calculated, contoured at 2σ (red). The Mn2+ atoms (purple) are depicted with surrounding residues (yellow sticks from domain1 and green sticks from domain2). (B) Stereoview of metal binding site with a magnesium ion in other subunit of the Mn2+-bound BhMntR. A σA-weighted electron density map (2Fo-Fc map) contoured at 1.0σ (blue). Anomalous map was calculated, contoured at 2σ (red). (C) Anomalous maps were calculated around metal binding site with binuclear manganese ions with different contour level (5σ, 4σ, and 3σ).

(TIF)

Click here for additional data file.

Structural comparisons of BhMntR with BsMntR.

(DOCX)

Click here for additional data file.

Apo BhMntR coordinate.

(PDB)

Click here for additional data file.

Apo BhMntR structure factor.

(MTZ)

Click here for additional data file.

Mn-bound BhMntR coordinate.

(PDB)

Click here for additional data file.

Mn-bound BhMntR structure factor.

(MTZ)

Click here for additional data file.

Validation report of apo BhMntR structure.

(PDF)

Click here for additional data file.

Validation report of Mn-bound BhMntR structure.

(PDF)

Click here for additional data file.

11 Oct 2019

PONE-D-19-24432

Structural analysis of the manganese transport regulator MntR from n class="Species">Bacillus halodurans in apo and manganese bound forms

PLOS ONE

Dear Dr. Jae Young Lee

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS On class="Chemical">NE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR: Please try to improve your manuscript according to the reviewers' criticism.

==============================

We would appreciate receiving your revised manuscript by Nov 25 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions n class="Chemical">Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Eugene A. Permyakov, Ph.D., Dr.Sci.

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS On class="Chemical">NE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We  found  text overlap between the current submission and your previously published works outside the Method section:

- https://www.nature.com/articles/s41598-018-31676-z

- http://scripts.iucr.org/cgi-bin/paper?S1399004714004118

- http://koreascience.or.kr/article/JAKO201425560113942.page

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed."

3.  We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This paper presents crystal structure data on a further member of the MntR family of magnesium transporter regulators. As expected, the structure is largely similar to previously reported structures. But there are some new details here regarding metal binding sites. The crystallographic work appears to be mainly in order, but with one issue noted below. I have one substantive critique of the analysis and some relatively minor comments and suggestions prior to publication.

Important concern:

Nothing is said by the authors about anomalous scattering. They might have generated stronger anomalous signal from the Mn had they collected data at a longer x-ray wavelength, and in view of this it’s not clear why the wavelength was chosen to be at 0.979 Ang (which is a good choice for n class="Chemical">selenium studies, not applicable here). Maybe the authors could get lucky and anomalous signal in a map would be detectable even at the sub-optimal wavelength where they collected the data (f’’ for Mn looks to be about 1.3 electrons at 0.979 Ang). The authors should calculate an anomalous difference map to see if the binuclear site shows up. If it does, then they could go on to look at the other, supposedly unoccupied, site. There they inferred a Mg ion, but as noted above it doesn’t make a lot of sense that a Mg would be there when it wasn’t there in the apo form. It’s conceivable that the density in the other site is a partially occupied Mn (not Mg). And if so maybe an anomalous map would give an indication. Having a better assignment of what’s in the other site would improve the findings of the paper.

Minor points:

1) There are some places where an editing for English usage will be important (e.g. recombination vs recombinant).

2) Lines 61-69 in the Introduction discuss some structural details of the MntR protein. As it is written is sounds like the authors might be describing results from the present study (which would be out of place in the Introduction), whereas they are presumably discussing what is known about the structure from prior work. This should be made clearer, e.g. “From previous structural studies it is understood that…”

3) The R-values are reported with a number of significant digits that seems one too many.

4) The legend to Fig 2 needs to explain better the distinction (in panels b AND c) between the site where the binuclear site is seen vs the n class="Chemical">manganese site.

5) The authors show 2Fo-Fc and omit (difference) maps to show the metal sites. This is ok. But they have the opportunity here to also calculate and show maps that are based on differences between the Fobs from the two data sets (i.e. Fobs(+metal) – Fobs(-metal), phased with a model without metals). This kind of map is a more direct examination of the observed differences and less model-dependent. The authors should examine this kind of map. Presumably it will show up the binuclear Mn cluster just as the Fo-Fc map did. But the authors might discern more about the unexpected n class="Chemical">magnesium site. Currently they say that no magnesium is seen in the ‘apo’ structure, despite the high concentration of Mg in the crystallization conditions. If Mg is really absent from that site in the apo form, then they should see a positive peak there in the Fo(+metal) – Fo(-metal) map.

Reviewer #2: This manuscript describes MntR (from Bacillus halodurans) structures in the apo and metal bound states. The assignment of n class="Chemical">Mn2+ and Mg2+ in the binding sites was not solid. It needs to be substantialized by additional measurements such as X-ray anomalous signals. The reported structures do not provide new biological insights in addition to the many existing MntR structures.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

18 Oct 2019

Dear Eugene A. Permyakov, Ph.D. Academic Editor, PLOS ONE

Reference code PONE-D-19-24432

TITLE: “Structural analysis of the manganese transport regulator MntR from n class="Species">Bacillus halodurans in apo and manganese bound forms”

Thank you very much for your kindly informing me of the reviewer’s comments on my manuscript submitted for a possible publication in the PLOS ONE. Please find the uploaded revised manuscript at the web address.

The revised manuscript takes into account all the comments made by the reviewers and editor. A separate list is attached to this letter to describe all the changes in detail.

Sincerely yours,

List of changes by reviewer 1

1. << Important concern: n class="Chemical">Nothing is said by the authors about anomalous scattering. They might have generated stronger anomalous signal from the Mn had they collected data at a longer x-ray wavelength, and in view of this it’s not clear why the wavelength was chosen to be at 0.979 Ang (which is a good choice for selenium studies, not applicable here). Maybe the authors could get lucky and anomalous signal in a map would be detectable even at the sub-optimal wavelength where they collected the data (f’’ for Mn looks to be about 1.3 electrons at 0.979 Ang). The authors should calculate an anomalous difference map to see if the binuclear site shows up. If it does, then they could go on to look at the other, supposedly unoccupied, site. There they inferred a Mg ion, but as noted above it doesn’t make a lot of sense that a Mg would be there when it wasn’t there in the apo form. It’s conceivable that the density in the other site is a partially occupied Mn (not Mg). And if so maybe an anomalous map would give an indication. Having a better assignment of what’s in the other site would improve the findings of the paper. >> - We calculated an anomalous difference map with Mn2+-bound BhMntR data and found the two clear peaks showing the manganese binuclear ions in metal binding site. The additional information was added on lines 200-202, page 10, stating that “We obtained Mn2+-bound crystals by soaking with 50 mM MnCl2 in apo crystals, and confirmed using an omit map and an anomalous map showing two peaks at the counter levels even at 5σ (Fig 2A and S2 Fig).”. In addition, the requested anomalous maps of Mn2+-bound BhMntR structures was added to supplemental figure (S2 Fig).

2. << There are some places where an editing for English usage will be important (e.g. recombination vs recombinant). >> - The English spelling was carefully checked and corrected on line 104, page 5, stating that “recombinant”.

3. << Lines 61-69 in the Introduction discuss some structural details of the MntR protein. As it is written is sounds like the authors might be describing results from the present study (which would be out of place in the Introduction), whereas they are presumably discussing what is known about the structure from prior work. This should be made clearer, e.g. “From previous structural studies it is understood that…” >> - The requested information was added as recommended on lines 62-69, page 3, stating that “From previous structural studies it is known that the metal binding site of B. subtilis MntR consists of several residues including Asp8 and Glu11 in domain 1, and His77, n class="Chemical">Glu99, Glu102 and His103 in domain 2.”

4. << The R-values are reported with a number of significant digits that seems one too many. >> - The R-n class="Chemical">values were corrected as recommended in text and table.

5. << The legend to Fig 2 needs to explain better the distinction (in panels b AND c) between the site where the binuclear site is seen vs the n class="Chemical">manganese site. >> - The original Fig2 legend sentence was changed on lines 212-217, page 10, stating that “(B) Metal binding site with binuclear manganese ions. The coordination with binuclear manganese ions and the distance between Mnc and the backbone carbonyl oxygen of Glu99 are shown in yellow and red, respectively. (C) Metal binding site with a magnesium ion. Unlike binuclear manganese ions binding, the magnesium ion (gray) forms a mononuclear cluster and the His77 made a hydrogen bond with Glu81 via a water (red).”

6. << The authors show 2Fo-Fc and omit (difference) maps to show the metal sites. This is ok. But they have the opportunity here to also calculate and show maps that are based on differences between the Fobs from the two data sets (i.e. Fobs(+metal) – Fobs(-metal), phased with a model without metals). This kind of map is a more direct examination of the observed differences and less model-dependent. The authors should examine this kind of map. Presumably it will show up the binuclear Mn cluster just as the Fo-Fc map did. But the authors might discern more about the unexpected n class="Chemical">magnesium site. Currently they say that no magnesium is seen in the ‘apo’ structure, despite the high concentration of Mg in the crystallization conditions. If Mg is really absent from that site in the apo form, then they should see a positive peak there in the Fo(+metal) – Fo(-metal) map.>> - We also calculated the Fo(+metal) – Fo(-metal) map. The two positive peaks were clearly shown around metal binding site indicating the binuclear manganese ions but no clear map was shown in magnesium binding site in the other subunit. In addition, the anomalous maps were calculated and shown in Supporting Information (S2 Fig).

List of changes by reviewer 2

1. << This manuscript describes MntR (from Bacillus halodurans) structures in the apo and metal bound states. The assignment of n class="Chemical">Mn2+ and Mg2+ in the binding sites was not solid. It needs to be substantialized by additional measurements such as X-ray anomalous signals. The reported structures do not provide new biological insights in addition to the many existing MntR structures. >> - We calculated an anomalous difference map with Mn2+-bound BhMntR data and found the two clear peaks showing the binuclear manganese ions in metal binding site. The additional information was added on lines 200-202, page 10, stating that “We obtained Mn2+-bound crystals by soaking with 50 mM MnCl2 in apo crystals, and confirmed using an omit map and an anomalous map showing two peaks at the counter levels even at 5σ (Fig 2A and S2 Fig).”. In addition, the requested anomalous maps of BhMntR structures was added to Supplementary figure (S2 Fig).

Journal Requirements:

1. << When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS On class="Chemical">NE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf >>. Our revised manuscript was generated with PLOS ONE style templates as requested.

2. << We found text overlap between the current submission and your previously published works outside the Method section:

- https://www.nature.com/articles/s41598-018-31676-z

- http://scripts.iucr.org/cgi-bin/paper?S1399004714004118

- http://koreascience.or.kr/article/JAKO201425560113942.page

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed." >> - We carefully checked text overlaps between the current submission and your previously published works outside the Method section. Main overlaps were found in Acknowledgement Section and the other minor overlap was rephrased on lines 291-293, page 13, stating that “Although the functional assignment of metal binding site for BhMntR is tentative, this structural model is applicable to other MntR homologous structures.”

3. << We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. >> - We already provided the accession numbers (6KTA and 6KTB) for PDB coordinates and structure factors and those data were included in Supporting Information.

Submitted filename: rebuttal_letter.docx

Click here for additional data file.

21 Oct 2019

Structural analysis of the manganese transport regulator MntR from n class="Species">Bacillus halodurans in apo and manganese bound forms

PONE-D-19-24432R1

Dear Dr. Jae Young Lee,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Eugene A. Permyakov, Ph.D., Dr.Sci.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

24 Oct 2019

PONE-D-19-24432R1

Structural analysis of the manganese transport regulator MntR from n class="Species">Bacillus halodurans in apo and manganese bound forms

Dear Dr. Lee:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Eugene A. Permyakov

Academic Editor

PLOS ONE