Diadenosine tetraphosphate (Ap4A) was discovered by Zamecnik in the middle 1960s.1 Ap4A is a product of the back reaction of the amino acid activation catalyzed by some aminoacyl-tRNA synthetases.2 It is widely agreed that there are aspects about the function of Ap4A. On the one hand, it may be an unavoidable by-product of protein synthesis that has to be cleared from the cell before it attains a potentially toxic concentration. On the other hand, Ap4A can function as a signal molecule and be deeply involved in regulation of DNA replication, cell division, and stress response.3
There are three kinds of Ap4A hydrolases, that is, asymmetrically cleaving Ap4A hydrolase (EC 126.96.36.199), symmetrically cleaving Ap4A hydrolase (EC 188.8.131.52), and dinucleoside polyphosphate phosphorylase (EC 184.108.40.206). Asymmetrical Ap4A hydrolases mainly exist in higher eukaryotes and play a key role in regulating the intracellular Ap4A level. Symmetrical Ap4A hydrolases have been isolated from many organisms, typically from bacteria. It catalyses the hydrolysis of Ap4A into two ADP and also hydrolyzes Ap5A, Gp4G, and other extending compounds. Furthermore, it is important in responding to heat shock and oxidative stress via regulating the concentration of Ap4A in bacteria.4
Here we report the crystal structure of the symmetrically cleaving Ap4A hydrolase from Shigella flexneri 2a which may cause bacillary dysentery or shigellosis in man.
Materials and Methods.
Cloning, Expression, and Purification.
The gene of Ap4A hydrolase from Shigella flexneri 2a str. 301 (Genbank: NC_004337) was cloned into the pET-22b(+) expression vector (Novagen). SeMet-substituted proteins were produced in E. coli BL21 (DE3). After harvested by centrifugation, the cells were resuspended in 30 mL of lysis buffer (25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10% glycerol, 5 mM β-Mercaptoethanol) and sonicated. The cell lysate was centrifugated at 12,000 rpm at 4°C for 30 min. Then the soluble fraction was applied to a Ni-NTA column (Amersham Pharmacia Biotech) preequilibrated with lysis buffer. The column was washed with wash buffer (25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 20 mM imidazole, 5 mM β-Mercaptoethanol). Finally the target protein was eluted with elution buffer (25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 200 mM imidazole, and 5 mM β-Mercaptoethanol). The eluate was applied to a 1-mL RESOURCE-Q anion exchange column (Amersham Pharmacia Biotech). The protein was eluted by using a linear gradient of 0–0.6M NaCl. The fraction with the target protein was concentrated to 1 mL, and was applied to HiLoad™ 16/60 Superdex 75 (prep grade) (Amersham Pharmacia Biotech), which was eluted with buffer consisting of 10 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 5 mM DTT.
Crystallization and Data Collection.
Crystals of SeMet- substituted Ap4A hydrolase were grown at 291 K by the hanging-drop vapor diffusion method. The reservoir solution was 100 mM Bis-Tris buffer (pH 6.8), containing 200 mM Magnesium chloride and 25% polyethylene glycol 3350. Protein solution (1 μL) with concentration of about 5 mg/mL was typically mixed with 1 μL reservoir solution and suspended on a cover slip over 0.45 mL reservoir solution. The crystals were picked up with Hampton mounting loops and frozen directly in liquid nitrogen. The crystal belongs to the space group C2, with unit cell dimensions a = 167.50 Å, b = 54.97 Å, c = 119.20 Å, and β = 129.14°. There are two molecules per asymmetric unit.
Structure Solution and Refinement.
X-ray diffraction data of the SeMet-substituted Ap4A hydrolase crystal were obtained on the beamline 3W1A at the Beijing Synchrotron Radiation Facility (BSRF), Institute of High Energy Physics, Chinese Academy of Sciences, using a MAR 165 mm CCD area detector at 100 K. Diffraction data were processed with the programs DENZO and SCALEPACK.5 The initial phasing was solved by SAD method6 using the program SOLVE.7 The initial model consisting of 30% of the Ap4A hydrolase structure was built by the program RESOLVE.8 The rest of the model was manually built using O.9 Refinement was carried out using the program REFMAC.10 The stereochemistry of the structure was checked by the program PROCHECK.11 The final model comprises 534 amino acid residues, 162 water molecules, and four manganese ions in the asymmetric unit. 24 amino acid residues at the C-terminal were not located, due to lack of electron density, and were excluded from the final model in the asymmetric unit. The statistic is summarized in Table I.
Table I. Data Collection, Phasing, and Refinement Statistics
The values in parentheses refer to data in the highest shell.
Rsym = Σ(|I| – |〈I〉|)/Σ|I|.
Rfree calculated using random 5% of total reflections omitted from refinement.
Unit cell parameters
a = 167.50 Å, b = 54.97 Å, c = 119.20 Å, β = 129.14°
Identification of the metal ion in the Ap4A hydrolase was carried out with an ICP optical emission spectrometer (ICP-OES). Purified protein was applied for the ICP-OES with protein concentration of about 0.3 mg/mL.
Results and Discussion.
The crystallographic asymmetric unit of the Ap4A hydrolase structure contains two molecules (A and B) that are related by a noncrystallographic twofold axis. The structures of two molecules are very similar, with root-mean-square (rms) deviation of 0.46 Å for all α carbon atoms [Fig. 1(A)]. The overall structure of Ap4A hydrolase monomer is shown in Figure 1(B). The Ap4A hydrolase molecule has dimensions of approximately 50 Å × 35 Å × 30 Å. The secondary structure elements, including 12 α-helices and 9 β-strands, are shown in Figure 1(B). The determined structure shows the existence of a narrow groove along the molecule surface, which is composed of α1, α2, α3, and α12. Based on the Fo–Fc density map and the result of the ICP-OES measurement, two manganese(II) ions separated by a distance of 3.45 Å have been found at the bottom of the groove. The dinuclear manganese cluster is bound to Asp8, His10, Asp37, Asn65, His120, His227, and four water molecules in the groove [Fig. 1(C)]. The six residues participating in binding to the dimanganese cluster are highly conserved in the protein family (Fig. 2).
Recently, the crystal structure of asymmetrical Ap4A hydrolase from Caenothabditis elegans (PDB ID 1KT91) was determined.14 Although symmetrical Ap4A hydrolase and asymmetrical Ap4A hydrolase share the same substrate Ap4A, the structural comparison between them indicates that the structure of symmetrical Ap4A hydrolase is not notably similar to that of asymmetrical Ap4A hydrolase. Using the DALI server15 (http://www.edi.ac.uk/dali/), the symmetrical Ap4A hydrolase structure was compared with the structures in the PDB database. The comparison results have revealed that the closest structural homologue is the bacteriophage λ Ser/Thr protein phosphatase (λPP, PDB ID 1G5B). This is also consistent with the results of conservation analysis by Barton et al.16 However, there is only 21% sequence identity between Ap4A hydrolase and λPP. The major differences between the two structures are located at the C-terminus where Ap4A hydrolase has six additional α-helices and an additional β-strand. λPP has a phosphatase active site with the sequence motif DXH(X)nGDXXDR(X)n GNHE and a dinuclear metal cluster.17 Most of the sequence motif and the dinuclear metal cluster are also present in symmetrical Ap4A hydrolase and homolog proteins [Figs. 1(C) and 2]. The structural comparison shows that Ap4A hydrolase has a spacial residue arrangement similar to the structure of λPP active site. The putative active site of symmetrical Ap4A hydrolase may resemble the λPP active site.
The crystal structure of the Ap4A hydrolase from Shigella flexneri 2a is the first structure determined for symmetrical Ap4A hydrolases. Its structure, in combination with further biochemical and biophysical studies, may reveal the structure–function relationship of symmetrically cleaving Ap4A hydrolases.
Diffraction data were collected at Beijing Synchrotron Radiation Facility (BSRF).
Protein Data Bank Accession Code. Coordinates and structure factors for the structure of Ap4A hydrolase have been deposited at the Protein Data Bank with ID code 2DFJ.