Overexpression, purification, crystallization and preliminary X-ray crystallographic characterization of the receiver domain of the response regulator PhoP from Enterococcus faecalis ATCC 29212
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Phosphate (Pho) regulon plays a critical role in bacterial phosphate homeostasis. It is regulated by two-component system (TCS) that comprises a sensor histidine kinase and transcriptional response regulator (RR). PhoP from Enterococcus faecalis (EfPhoP) belongs to the OmpR subfamily of RRs. It has not yet been structurally characterized because it is difficult to crystallize it to full-length form. In this study, a truncated form of EfPhoP containing the receiver domain (EfPhoP-RD) was constructed, purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. The crystal of EfPhoP-RD diffracted to 3.5 Å resolution and belonged to the orthorhombic space group C2221, with unit-cell parameters a = 118.74, b = 189.83, c = 189.88 Å. The asymmetric unit contains approximately 12 molecules, corresponding to a Matthews coefficient (Vm) of 2.50 Å3 Da−1 with a solvent content of 50.9%.
KeywordsPhosphate (Pho) regulon Two-component system Response regulator PhoP Enterococcus faecalis
Phosphorous is one of cellular component important for many biological and biochemical processes in living organisms, such as the formations of nucleic acids (DNA and RNA) and membrane phospholipids, post-translational modifications for signal transduction, etc. . The usefulness of phosphorous has been applied to extensive field such as agriculture, medicine and pharmaceuticals. In particular, phosphorous is required to secure a high level of productivity in agriculture [2, 3]. In bacteria, the major form of phosphorous is orthophosphate known as inorganic phosphates (Pi) . Despite the varying importance of Pi in cellular function, it is usually found at very low concentration in natural environment . Therefore, bacteria and other organisms must have relevant systems that include physiological and biochemical responses to overcome the deficiency of this nutrient .
A unique mechanism associated with the maintenance of Pi in bacteria as a regulatory circuit is the phosphate (Pho) regulon that is regulated by a two-component system (TCS) [5, 6]. The Pho regulon is one of the most rational and effective regulatory mechanisms. It is well-studied in model cells such as Escherichia coli  and Bacillus subtilis . Later, it has been characterized in many other bacterial species . TCSs are signal transduction pathways commonly used by bacteria to recognize and adapt to stimuli caused by environmental changes. TCS consists of histidine kinase (HK) as an inner-membrane sensor kinase that recognizes several specific environmental signals and the transcriptional response regulator (RR) protein that mediates cellular responses by regulating expression of specific genes or modulating protein functions in the cytoplasm . Although these proteins are known by different names in some bacteria [11, 12], upon Pi deficiency, the RR is phosphorylated by the HK. Thereby, the phosphorylated RR can bind to specific DNA sequences and then activate or suppress the transcription of their corresponding genes [13, 14].
A number of new members of Pho regulon have been identified from several bacteria in past years, but there still remain numerous undiscovered questions such as the detailed function of the entire system and the mechanisms connecting the Pho regulon to pathogenesis . Among them, enterococci are normal flora in human intestine of healthy adults, but also they are one of the major causes of hospital infections that leads a variety of diseases, including bacteremia, urinary tract and central nervous system infections . Most clinical isolates of enterococci are Enterococcus faecalis along with Enterococcus faecium . However, the TCS related to the Pho regulon in this strain has not been well studied except for the VanRS system that regulates the resistance of enterococci to vancomycin .
Most RRs have two distinct domains involving the receiver domain of N-terminus and the effector domain of C-terminus. On the basis of the structure and function of the effector domains, they can be classified into subfamilies . PhoP belongs to the OmpR/PhoB subfamily of RRs, including the OmpR and PhoB as the representative members . To date, only a few structures of PhoP have been reported because it is difficult to crystallize the full length of RRs in this subfamily . Thus, more detailed structure information is required to understand their functional mechanisms such as the conformational changes accompanying with phosphorylation of PhoP and to compare with known PhoP structures. To determine its structure, a truncated form of PhoP from E. faecalis containing the receiver domain (EfPhoP-RD) was constructed as the first step. Here, we report the crystallization conditions and preliminary X-ray crystallographic analysis of EfPhoP-RD. Complete diffraction data sets was collected from apo-crystals at resolutions up to 3.5 Å.
Materials and methods
Overexpression and purification of EfPhoP-RD protein
Enterococcus faecalis ATCC 29212
5′-GCG GCG CAT ATG AAA AAA GTT CTT GTC GTC-3′
5′-CTA CTA CTC GAG CTC TTG AAG CGT TTC GGT-3′
E. coli BL21 (DE3)
Complete amino-acid sequence
of the construct producedb
The harvested cell pellets were resuspended in pre-equilibrium buffer A (0.02 M Tris–HCl, pH 7.5, 0.5 M NaCl, 10% glycerol) adding 1 mM phenylmethylsulfonyl fluoride and ruptured by ultrasonication at 4 °C. The crude lysate was centrifuged at 25,000g for 20 min at 4 °C. The supernatant was loaded onto a nickel (Ni2+) charged HisTrap HP column (GE Healthcare, USA) equilibrated in buffer A. The bound EfPhoP-RD on the column was eluted with a linear gradient of elution buffer containing 0.02 M Tris–HCl, pH 7.5, 0.5 M imidazole, 0.5 M NaCl, 10% glycerol. The collected each fraction was confirmed by 15% SDS-PAGE, and subsequently purified by size exclusion chromatography on a HiLoad Superdex 200 column (GE Healthcare, USA) pre-equilibrated with buffer containing 0.02 M Tris–HCl pH 7.5, 0.15 M NaCl, 10% glycerol. The collected fractions containing EfPhoP-RD were pooled and concentrated to 7.4 mg/ml using an Amicon Ultra-15 centrifugal filter device (Millipore, USA).
Preliminary screening for the crystallization of EfPhoP-RD was performed by the hanging-drop vapour-diffusion method in 96-well microplates at 21 °C using various commercial screening kits such as Crystal Screen 1 and 2, PEGRx 1 and 2 (Hampton Research, USA), and Wizard Classic 1, 2, 3 and 4 (Rigaku Reagents Inc., USA). Initial crystals were obtained from two solutions as follows: the condition No. 30 of Crystal Screen 2 [0.1 M HEPES, pH 7.5, 5% (v/v) (+/−)-2-methyl-2,4-pentanediol, 10% (w/v) polyethylene glycol (PEG) 6000] and the condition No. 19 of PEGRx 2 [0.1 M Bis–Tris–propane, pH 9.0, 0.1 M NaCl, 25% (w/v) PEG 1500]. Optimization of EfPhoP-RD crystal was performed with the hanging-drop vapour-diffusion method in 24-well VDX plates (Hampton Research, USA) under conditions containing various PEGs and pH ranges; Each hanging drop was made by adding 1 μl protein solution to 1 μl reservoir solution to be a total volume of 2 μl and was then equilibrated against 500 μl reservoir solution.
Collection and analysis of X-ray diffraction data
Beamline 7A, PAL
ADSC Quantum 270r CCD
Crystal-to-detector distance (mm)
Rotation range per image (°)
Total rotation range (°)
Exposure time per image (s)
Unit-cell parameters (Å, °)
a = 188.63, b = 187.71, c = 197.49
α = β = γ = 90
a = 118.74, b = 189.83, c = 189.88
α = β = γ = 90
Resolution range (Å)
Total no. of reflections
No. of unique reflections
Results and discussion
Molecular replacement was attempted using MOLREP program  in the CCP4  with the crystal structure of BsPhoP-RD (PDB ID: 1MVO)  indicating 54% sequence identity as a search model. However, our attempts could not provide a clear solution of structure for further refinement. This implies that the structure of EfPhoP-RD might contain a novel or different fold compared to other PhoP-RDs, although all data sets had low completeness and resolution. Therefore, the structure of EfPhoP-RD will be further determined by the MAD method  using selenomethionine substituted protein to solve the phase problem.
We would like to thank the staff of beamline 7A at the Pohang Accelerator Laboratory in South Korea for their assistance during X-ray data collection.
YCJ and KSL carried out experiments. YCJ and KSL designed experiments, analyzed data and wrote the manuscript. Both authors read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
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