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

Summary

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.

Keywords:

Protein domains – Crystal structure – Manganese – Hydrogen bonding – Glycerol – Crystals – Dimerization – magnesium

Introduction

Metal ions are essential for living organisms because iron, zinc, and 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 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 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], 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).

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 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 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 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 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) 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.

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 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 B. subtilis (2HYG). The structure was refined to crystallographic R_work and R_free 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 R_work value of 17.1% and an R_free 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 Ų, 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 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 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 Ų and 63.88 Ų, 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).

The binuclear manganese ions were liganded by six amino acid residues: Asp8 and Glu11 contributed by domain 1, and His 77, Glu99, Glu102, and His103 contributed by domain 2 (Fig 2B). The two manganese ions (Mn_A and Mn_C) were jointly coordinated by the carboxylate oxygens of Glu99 and Glu102 from domain 2. Each metal ion was individually coordinated by Glu11 (Mn_A), His77 (Mn_A), His103 (Mn_C) and Asp8 (Mn_C). The Mn_A 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 Mn_c 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 Mn_c 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 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 Mn_A 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 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 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.

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 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 Mn_C 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.

Supporting information

S1 Fig [tif]
R.m.s.d plot of MntR.

S2 Fig [a]
Anomalous maps in metal ion binding site of MntR.

S1 Table [docx]
Structural comparisons of MntR with MntR.

S1 File [pdb]
Apo MntR coordinate.

S2 File [mtz]
Apo MntR structure factor.

S3 File [pdb]
Mn-bound MntR coordinate.

S4 File [mtz]
Mn-bound MntR structure factor.

S5 File [pdf]
Validation report of apo MntR structure.

S6 File [pdf]
Validation report of Mn-bound MntR structure.