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Cobalt complex structure of the sirohydrochlorin chelatase SirB from Bacillus subtilis subsp. spizizenii
Korean J. Microbiol. 2019;55(2):123-130
Published online June 30, 2019
© 2019 The Microbiological Society of Korea.

Mi Sun Nam, Wan Seok Song, Sun Cheol Park, and Sung-il Yoon*

Division of Biomedical Convergence, College of Biomedical Science, Kangwon National University, Chuncheon 24341, Republic of Korea
Correspondence to: E-mail: sungil@kangwon.ac.kr; Tel.: +82-33-250-8385; Fax: +82-33-259-5643
Received April 22, 2019; Revised June 5, 2019; Accepted June 5, 2019.
Abstract

Chelatase catalyzes the insertion of divalent metal into tetrapyrrole and plays a key role in the biosynthesis of metallated tetrapyrroles, such as cobalamin, siroheme, heme, and chlorophyll. SirB is a sirohydrochlorin (SHC) chelatase that generates cobalt-SHC or iron-SHC by inserting cobalt or iron into the center of sirohydrochlorin tetrapyrrole. To provide structural insights into the metal-binding and SHC-recognition mechanisms of SirB, we determined the crystal structure of SirB from Bacillus subtilis subsp. spizizenii (bssSirB) in complex with cobalt ions. bssSirB forms a monomeric α/β structure that consists of two domains, an N-terminal domain (NTD) and a C-terminal domain (CTD). The NTD and CTD of bssSirB adopt similar structures with a four-stranded β-sheet that is decorated by α-helices. bssSirB presents a highly conserved cavity that is generated between the NTD and CTD and interacts with a cobalt ion on top of the cavity using two histidine residues of the NTD. Moreover, our comparative structural analysis suggests that bssSirB would accommodate an SHC molecule into the interdomain cavity. Based on these structural findings, we propose that the cavity of bssSirB functions as the active site where cobalt insertion into SHC occurs.

Keywords : chelatase, cobalt, crystal structure, SirB, sirohydrochlorin
Body

Metallated tetrapyrroles, including cobalamin, coenzyme F430, siroheme, heme, and chlorophyll, function as essential cofactors of diverse proteins and drive numerous biological processes, such as metabolism, photosynthesis, and oxygen transport (Raux et al., 2000; Schubert et al., 2002). To generate functional metallated tetrapyrroles, divalent metals, such as cobalt, iron, magnesium, and nickel, should be inserted into the center of tetrapyrroles through the enzymatic activity of chelatase. Chelatases fall into three classes (I, II, and III) based on ATP dependence and additional catalytic activity (Brindley et al., 2003). Class I and class II chelatases are relatively well characterized through structural and biophysicochemical studies. Class I includes ATP-dependent magnesium or cobalt chelatases that consist of three subunits (ChlH/I/D or CobN/S/T) (Debussche et al., 1992; Walker and Willows, 1997). Class II chelatases exist as a monomer or a homocomplex and include CbiX, CbiK, and SirB, which insert cobalt or iron into SHC in an ATP-independent manner (Al-Karadaghi et al., 1997; Schubert et al., 1999; Leech et al., 2002, 2003; Raux et al., 2003; Yin et al., 2006; Romao et al., 2011; Fujishiro et al., 2019).

In the Archaea, CbiX exists as a small enzyme that consists of 120–145 residues and is regarded as the simplest form of CbiX (CbiXS) (Brindley et al., 2003). A structural analysis of Archaeoglobus fulgidus CbiXS (afCbiXS) revealed that afCbiXS forms a dimer that accommodates a cobalt-SHC molecule into an intersubunit cavity (Yin et al., 2006; Romao et al., 2011). In contrast to the single domain structure of CbiXS, CbiK is composed of two domains, each of which resembles CbiXS in size and overall structure (Schubert et al., 1999; Romao et al., 2011; Bali et al., 2014; Lobo et al., 2017). Therefore, CbiXS is considered as an ancestral form of class II chelatases.

Due to extensive structural studies on CbiXS and CbiK, the structural features and substrate-recognition mode of CbiXS and CbiK have been well characterized. However, the three-dimensional structure of SirB has been elusive until recently. In 2019, the crystal structure of Bacillus subtilis strain 168 SirB (bs168SirB) was reported (Fujishiro et al., 2019). Despite the biological significance of SirB in the biosynthesis of metallated tetrapyrroles, only bs168SirB has been structurally defined to date. Thus, it is unclear whether the structural features of bs168SirB are applied to other SirB proteins. To further characterize the interaction of SirB with metal and provide structural insights into the active site of SirB, we determined the crystal structure of SirB from B. subtilis subsp. spizizenii (bssSirB) in complex with cobalt ions. Based on a comparative structural analysis of SirB, CbiXS, and CbiK, we provide a unique cobalt-binding mechanism of SirB and propose that SirB accommodates an SHC molecule into an interdomain cavity.

Materials and Methods

Construction of protein expression vector

bssSirB-encoding DNA fragment was amplified by PCR using the genomic DNA of B. subtilis subsp. spizizenii strain W23 with DNA primers (forward primer, 5’-TAAGGATCCGATGAAGCAAGCAATTTTATATGTCGGTC-3’; reverse primer, 5’-GCCGATGTCGACCTAATGTGCAGCGGGAGCATATGAACC-3’) containing the recognition site of either BamHI or SalI restriction enzyme. The PCR-amplified DNA was digested by the BamHI and SalI restriction enzymes and inserted into a pET49b vector that was modified to express recombinant protein in N-terminal fusion with a hexa-histidine tag and a thrombin cleavage site (Park et al., 2017). The ligated product was transformed into Escherichia coli DH5α cells, and transformants were selected by kanamycin. The nucleotide sequence of the bssSirB-encoding region in the expression vector was verified by DNA sequencing.

Protein expression and purification

To express recombinant bssSirB protein, bssSirB expression vector was transformed into E. coli BL21 (DE3) cells, and the transformant cells containing bssSirB expression vector was selected in the presence of kanamycin. The cells were first grown in LB medium at 37°C. When the optical density of the culture at 600 nm reached 0.6~0.8, IPTG was added into the culture to the final concentration of 1 mM for the induction of protein expression. The cells were further cultured overnight at 18°C and harvested by centrifugation.

bssSirB protein-containing cells were lysed by sonication in a solution containing 50 mM Tris (pH 8.0), 200 mM NaCl, 5 mM β-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride. The cell lysate was cleared by centrifugation, and the resultant supernatant was incubated with Ni-NTA resin at 4°C. The resin was harvested using an Econo-Column (Bio-Rad) and washed using a solution containing 50 mM Tris (pH 8.0), 200 mM NaCl, 5 mM β-mercaptoethanol, and 10 mM imidazole. bssSirB protein was eluted by the stepwise increase of imidazole concentration (20 mM, 50 mM, and 250 mM). bssSirB protein was mainly eluted in a solution containing 50 mM Tris (pH 8.0), 200 mM NaCl, 5 mM β-mercaptoethanol, and 250 mM imidazole. bssSirB protein was dialyzed against 20 mM Tris (pH 8.0) and 5 mM β-mercaptoethanol and subjected to thrombin digestion at 18°C for 3.5 h to remove the N-terminal hexa-histidine tag. The resulting tag-free bssSirB protein was further purified by anion exchange chromatography using a Mono Q 10/100 column with a gradient of 0~500 mM NaCl in 20 mM Tris (pH 8.0) and 5 mM β-mercaptoethanol. The purified bssSirB protein was concentrated to ~13 mg/ml for crystallization.

Crystallization and X-ray diffraction

bssSirB protein was crystallized by a sitting-drop vapor-diffusion method at 18°C in a drop containing 0.5 µl of ~13 mg/ml bssSirB protein and 0.5 µl of reservoir solution. Initial crystals of bssSirB were obtained in the JCSG Core Suite kit (Qiagen) using a reservoir solution containing 10 mM cobalt chloride, 1.8 M ammonium sulfate, 0.1 M MES (pH 6.5). The initial bssSirB crystallization condition was optimized to 10 mM cobalt chloride, 0.1 M magnesium chloride, 1.3 M ammonium sulfate, and 0.1 M MES (pH 6.5).

X-ray diffraction of bssSirB crystals was performed at beamline 7A, the Pohang Accelerator Laboratory (Republic of Korea). A single bssSirB crystal was transferred to a cryoprotectant solution containing 10 mM cobalt chloride, 0.1 M magnesium chloride, 1.4 M ammonium sulfate, 0.1 M MES (pH 6.5), and 25% glycerol, and the crystal was mounted on a goniometer using a nylon loop under a cryo-stream at -173°C. X-ray with a wavelength of 1.00004 Å was applied to the mounted crystal, and X-ray diffraction data were collected using an ADSC Quantum 270 detector. The full dataset was indexed, integrated, scaled, and merged using the HKL2000 program (Otwinowski and Minor, 1997).

Structure determination

The crystal structure of bssSirB was determined by molecular replacement with the Phaser program (version 2.1.4) using the bs168SirB structure (PDB ID 5ZT7) as a search model (McCoy et al., 2007; Fujishiro et al., 2019). The molecular replacement solution was modified to the final structure of bssSirB through iterative cycles of model building and refinement using the Coot program (version 0.6.2) and the Refmac5 program (version 5.5.0109), respectively (Murshudov et al., 1997; Emsley and Cowtan, 2004). The structures of bssSirB and its homologs were analyzed using the CCP4 suite (version 2.0.6) and the Coot program (version 0.6.2) (Emsley and Cowtan, 2004; Winn et al., 2011). The structures were visualized by the PyMOL program (version 1.4.1) (www.pymol.org).

Accession number

The atomic coordinates and the structure factors for bssSirB (PDB ID 6JV6) have been deposited in the Protein Data Bank (www.rcsb.org).

Results and Discussion

Overall structure of bssSirB

As a first step to determine the crystal structure of bssSirB, recombinant bssSirB protein was expressed in E. coli cells and purified by Ni-NTA affinity chromatography and anion exchange chromatography. SDS-PAGE and gel-filtration chromatography analyses showed that purified bssSirB protein is monodisperse in protein size and oligomeric state (Fig. 1). Moreover, bssSirB whose molecular weight is 29.3 kDa was eluted between 17 kDa and 44 kDa protein standards, indicating that bssSirB is monomeric in solution (Fig. 1C).

Fig. 1.

Sequence and biochemical characterization of bssSirB. (A) Amino acid sequence and secondary structure elements (α-helix, wave; β-strand, arrow) of bssSirB. The residues that were built in the bssSirB structure are indicated by lines above the sequence. The secondary structure elements of the NTD and CTD of bssSirB are labeled in black and blue, respectively. (B) SDS-PAGE analysis of purified bssSirB protein. (C) Gel-filtration chromatography analysis of purified bssSirB protein.



bssSirB protein was crystallized in a cobalt-containing solution, and a bssSirB crystal diffracted X-ray to 2.15 Å resolution. The crystal structure of bssSirB was determined by molecular replacement and refined to an Rfree value of 24.8% (Fig. 2A and Table 1). To be consistent with a monomeric form of bssSirB in gel-filtration chromatography, only one bssSirB polypeptide chain was identified in the asymmetric unit of the bssSirB crystal. The bssSirB structure contains residues 1–120, 127–134, 136–165, 171–179, and 186–252 from the entire polypeptide chain of bssSirB that consists of 261 residues.

Fig. 2.

Crystal structure of bssSirB. (A) Overall structure of bssSirB. bssSirB is depicted as a rainbow ribbon representation (N-terminus, blue; C-terminus, red). Two cobalt ions are shown as salmon spheres. (B) Similar structures of the NTD and CTD of bssSirB. The structure of the bssSirB CTD (magenta ribbons) is overlaid on that of the bssSirB NTD (green ribbons).


Crystallographic statistics of the bssSirB structure

Data collection & refinement bssSirB
Data collection
Space group P3221
Cell parameters a = b = 75.67 Å
c = 82.42 Å
Wavelength (Å) 1.00004
Resolution (Å) 30.00-2.15
Highest resolution (Å) 2.23-2.15
No. unique reflections 15,502
Rmerge (%)a 7.1 (50.2)b
I/sigma(I) 33.0 (4.5)b
Completeness (%) 99.4 (99.8)b
Redundancy 4.2 (4.3)b
Refinement
Resolution (Å) 30.00-2.15
No. of reflections (work) 14,420
No. of reflections (test) 726
Rwork (%)c 20.4
Rfree (%)d 24.8
No. atoms 1,816
Protein 1,769
Ligand (cobalt) 2
Water 45
Average B-value (Å2) 40.3
RMSD bonds (Å) 0.014
RMSD angles (°) 1.35
Ramachandrane (favored) 96.9%
(outliers) 0.0%
Rmerge = ΣhklΣi | Ii(hkl) - <I(hkl)> | / ΣhklΣi Ii(hkl)
Numbers in parenthesis were calculated from data of the highest resolution shell.
Rwork = Σ| |Fobs|-|Fcalc| | / Σ|Fobs|, where Fcalc and Fobs are the calculated and observed structure factor amplitudes, respectively.
Rfree = as for Rwork, but for 5% of the total reflections chosen at random and omitted from refinement.
Calculated using MolProbity (http://molprobity.biochem.duke.edu).


bssSirB is composed of two domains, an N-terminal domain (NTD) and a C-terminal domain (CTD) (Fig. 2A). The NTD covers residues 1–104 and 228–261, and the CTD includes residues 105–227. The two domains exhibit identical organization of β-strands and α-helices with alternating arrangements of a β-strand and an α-helix from the N-terminus to the C-terminus. In both NTD and CTD, four parallel β-strands form one β-sheet, which is decorated by two α-helices on each side. The CTD of bssSirB is superimposable on the NTD of bssSirB with a root-mean-square-deviation (RMSD) value of ~2.2 Å for 78 Cα atoms and ~17% sequence identity (Fig. 2B).

Cobalt binding by bssSirB

In the crystal structure of bssSirB, two high electron density peaks that cannot be explained by water molecules were identified and were built as cobalt ions (Co1 and Co2) because of inclusion of cobalt ions in the bssSirB crystallization solution (Fig. 3). The Co1 and Co2 ions are coordinated by residues from one bssSirB chain and its symmetry-related chain (bssSirB′). The Co1 cobalt ion is liganded by two histidine residues of bssSirB (H10 and H76) and one histidine residue of bssSirB′ (H31′) and is further coordinated by three water molecules, exhibiting an octahedral coordination (Zheng et al., 2014). The Co2 cobalt ion is coordinated by the H79 and E83 residues of bssSirB and two histidine residues of bssSirB′ (H115′ and H229′). Thus, cobalt ions appear to contribute to bssSirB crystallization by generating cobalt-mediated crystal contacts.

Fig. 3.

Cobalt ions in the crystal structure of bssSirB. (A) Two cobalt ions between bssSirB chain (green ribbons) and its symmetry-related chain (yellow ribbons). Cobalt ions (Co1 and Co2) are shown as salmon spheres. The cobalt-coordinating residues of bssSirB chain and its symmetry-related chain are shown as cyan and orange sticks, respectively. Cobalt-coordinating water molecules are depicted as small red spheres. (B) Sequence conservation of cobalt-coordinating residues from bssSirB chain and its symmetry-related chain. Sequence conservation is color coded by magenta intensity (high sequence conservation, magenta; low sequence conservation, white).



Active site of bssSirB

Protein residues that constitute the active site of enzyme are evolutionally conserved because the active site plays a key role in catalytic activity. To identify the active site of bssSirB, the sequence conservation of each bssSirB residue was calculated using the sequences of SirB orthologs and is shown on the molecular surface of bssSirB (Fig. 4A). An interdomain cavity between the NTD and CTD and its surrounding regions present high sequence conservation. Notably, Co1 is found on top of the cavity of bssSirB and is coordinated by the two histidine residues of the NTD (H10 and H76) that are absolutely conserved in SirB orthologs, suggesting that SirB uses the two histidine residues, corresponding to bssSirB H10 and H76, to coordinate a cobalt ion in solution (Figs. 3B and 4B). Moreover, when the structure of an afCbiXS dimer in complex with a cobalt-SHC molecule is overlaid on the bssSirB structure, the SHC molecule is found in the cavity of bssSirB (Fig. 4C) (Romao et al., 2011). These observations suggest that the interdomain cavity of bssSirB would accommodate a cobalt ion and a SHC molecule as a catalytic site to facilitate the transfer of the cobalt ion to SHC.

Fig. 4.

Sequence conservation and active site of bssSirB. (A) High sequence conservation of the cobalt-coordinating residues and cavity residues of bssSirB. Sequence conservation is color coded by magenta intensity (high sequence conservation, magenta; low sequence conservation, white) on the molecular surface of bssSirB. (B) Cobalt ion (Co1; salmon sphere) and its coordination by two bssSirB histidine residues (H10 and H76; green sticks) and three water molecules (small red spheres) in the bssSirB structure. (C) Tentative active site of bssSirB in the boxed region of Fig. 4A. The afCbiXS structure was superimposed on the bssSirB structure, and the cobalt-SHC molecule of the afCbiXS structure (PDB ID 2XWQ) is shown in the figure to present the tentative SHC-binding site of bssSirB.



Mutually exclusive cobalt-binding sites of SirB and CbiK

Each domain of bssSirB mimics afCbiXS in the organization of secondary structures with a helix-decorated β-sheet structure, and the overall structure of bssSirB resembles that of an afCbiXS dimer (Fig. 5A) (Romao et al., 2011). CbiK is also similar to the afCbiXS dimer in dimer organization and domain structure (Romao et al., 2011; Lobo et al., 2017). Based on this structural resemblance, we propose that the SirB and CbiK genes were generated through the duplication of the CbiXS gene and the subsequent fusion of the duplicated CbiXS genes. Although the overall structure of SirB is similar to that of CbiK, SirB and CbiK seem to have followed different evolutionary paths, given that they accommodate a cobalt ion using different domains (Fig. 5A). SirB employs two histidine residues of the NTD (H10 and H76 in bssSirB) to coordinate a cobalt ion above SHC, whereas CbiK employs two histidine residues [H154 and H216 in Desulfovibrio vulgaris CbiK (dvCbiK)] from the CTD below SHC. However, in bssSirB, dvCbiK H216 is replaced with a leucine residue (L200) that cannot coordinate a cobalt ion. In dvCbiK, bssSirB H10 is replaced with a phenylalanine residue (F17) that is incompatible with cobalt coordination. Thus, we propose that SirB and CbiK evolved to contain a mutually exclusive cobalt-binding site either in the NTD or the CTD by changing the other cobalt-binding site.

Fig. 5.

Comparative structural analysis of bssSirB and its homologs. (A) Similar structures and different cobalt coordination of SirB, CbiK, and CbiXS. The structure (PDB ID 2XVZ) of dvCbiK (yellow ribbons) complexed with cobalt (yellow sphere) and the structure (PDB ID 2XWQ) of afCbiXS dimer (magenta ribbons) complexed with cobalt-SHC (cyan sticks) are overlaid on the structure of bssSirB (green ribbons) in complex with cobalt (green sphere). In the inset, the cobalt-coordinating histidine residues (underlined and in bold) and their equivalent residues of bssSirB (green) and dvCbiK (yellow) are depicted as sticks, and only the cobalt-pyrrole moiety (cyan sticks) of the cobalt-SHC molecule from the afCbiXS structure is shown for clarity. The tentative direction of cobalt insertion into SHC is indicated by arrows (bssSirB, green; dvCbiK, yellow). (B) Sequence differences between bssSirB and bs168SirB. The bs168SirB structure (light blue lines; PDB ID 5ZT8) is overlaid on the bssSirB structure (green lines), and their different residues are represented by sticks (bssSirB, green; bs168SirB, light blue). The Co1 cobalt ion in the bssSirB structure is depicted as a sphere, and a tentative active site of bssSirB is highlighted by a dashed ellipse.



Structural comparison of bssSirB and bs168SirB

bssSirB shares 94% sequence identity with bs168SirB and exhibits high structural similarity to bs168SirB with an RMSD value of 0.72 Å despite different crystal contacts (space group of the bssSirB structure, P3221; space group of the bs168SirB structure, P21) (Fig. 5B) (Fujishiro et al., 2019). Moreover, in both bssSirB and bs168SirB structures, a cobalt ion was identified on top of the cavity and coordinated by the H10 and H76 residues. The 16 residues of bs168SirB that differ from those of bssSirB are located away from the cavity, suggesting that the sequence changes do not directly affect the enzyme activity of SirB (Fig. 5B).

적 요

Chelatase는 tetrapyrrole에 2가 금속을 삽입하는 데 관여하는 효소로서 cobalamin, siroheme, heme, chlorophyll과 같은 금속-tetrapyrrole의 생합성에 필수적인 역할을 담당한다. SirB는 sirohydrochlorin(SHC) tetrapyrrole의 중앙부에 코발트나 철을 삽입하여 코발트-SHC 또는 철-SHC를 형성하는 SHC chelatase이다. SirB의 금속 결합 기전 및 SHC 인식 기전을 구조적으로 이해하기 위해 Bacillus subtilis subsp. spizizenii에서 유래한 SirB(bssSirB)의 코발트 복합체 구조를 규명하였다. bssSirB는 N-말단 도메인(NTD)과 C-말단 도메인(CTD)으로 구성된 α/β 단량체 구조를 형성한다. bssSirB는 NTD와 CTD 사이에 서열 보존성이 높은 공동을 지니며 NTD의 histidine 잔기 2개를 이용하여 공동 상단에서 코발트 이온과 상호작용한다. 또한 구조 비교 분석 결과 bssSirB는 공동 내에 SHC 분자를 수용하는 것으로 판단된다. 이러한 구조적 발견에 기초하여 bssSirB의 공동은 SHC의 코발트 삽입이 이뤄지는 활성 부위임을 제안한다.

Acknowledgements

We express many thanks to staff members at beamline 7A, the Pohang Accelerator Laboratory (Republic of Korea) for help with X-ray diffraction. Diffraction of bssSirB crystals was performed at beamline 7A, the Pohang Accelerator Laboratory (Republic of Korea). This research was supported by a grant from the National Research Foundation of Korea (2016R1D1A1B03930540 to SIY) and by 2017 Research Grant from Kangwon National University (520170410 to SIY). There is no conflict of interest to declare.

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