Identification and characterization of the biochemical function of Agrobacterium T-complex-recruiting protein Atu5117

Authors


Abstract

Atu5117 from Agrobacterium tumefaciens is a highly conserved protein with a putative nucleotidyltransferase domain in its N-terminal region and a putative higher eukaryotes and prokaryotes nucleotide-binding domain in its C-terminal region. This protein has been shown to be a T-complex-recruiting protein that can recruit T-complex from the cytosol to the polar VirB/D4 type IV secretion system (T4SS). However, the biochemical function of Atu5117 is still unknown. Here, we show that Atu5117 is a (d)NTPase. Although no proteins with nucleotidyltransferase and higher eukaryotes and prokaryotes nucleotide-binding domains were identified as (d)NTPases, Atu5117 was able to convert all eight canonical NTPs and dNTPs to NDP, dNDP and inorganic phosphate in vitro, and required Mg2+ for its (d)NTPase activity. The kinetic parameters of Atu5117 (d)NTPase for eight substrates were characterized. Kinetic data showed that Atu5117 (d)NTPase preferred ATP as its substrate. The optimal conditions for (d)NTPase activity of Atu5117 were very similar to those required for Agrobacterium tumorigenesis. The kinetic parameters of (d)NTPase of Atu5117 for all four canonical NTPs were in the same orders of magnitude as the kinetic parameters of the ATPases identified in some components of the VirB/D4 T4SS. These results suggest that Atu5117 might function as an energizer to recruit T-complex to the T4SS transport site.

Abbreviations
HEPN

higher eukaryotes and prokaryotes nucleotide-binding

T4SS

type IV secretion system

TNP-ATP

3′(2′)-O-(2,4,6-trinitrophenyl)-ATP

VBP

VirD2-binding protein

Introduction

The Agrobacterium tumefaciens Atu5117 gene encodes a protein with 313 amino acids. This protein was annotated as a hypothetical protein in the Atumefaciens genome database. As well as Atu5117, A. tumefaciens has two additional genes (Atu4860 and Atu4856) encoding proteins that are highly similar to the Atu5117-encoded protein in amino acid sequence, but both Atu4860 and Atu4856 were annotated as highly conserved proteins in the database. Our previous investigation showed that Atu5117 (including its paralogous proteins, Atu4860 and Atu4856) can specifically bind to VirD2, and are involved in tumorigenesis; thus, these proteins were originally called VirD2-binding proteins (VBPs) [1]. Further study of the biological function of VBPs demonstrated that VBPs are involved in the recruitment of T-complex to the VirB/D4 type IV secretion system (T4SS) transport site [2]. Meanwhile, VBPs were also verified to be important in the recruitment of a conjugative plasmid to a different transfer system from the VirB/D4 T4SS. Therefore, VBPs were defined as ‘recruiting proteins’, a previously uncharacterized class of proteins involved in the recruitment of nucleoprotein substrate complex to the transport apparatus [2-4].

However, it remains obscure how the T-complex-recruiting protein carries out the recruiting function. A conserved domain search predicted that VBPs would contain a putative nucleotidyltransferase domain in their N-terminal region and a putative higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain in their C-terminal region [1]. Nucleotidyltransferases constitute a superfamily of enzymes catalyzing the transfer of a nucleotide to an acceptor hydroxyl group, and are currently classified into 83 enzyme members by the International Union of Biochemistry and Molecular Biology website (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/7/7/), depending on their distinct substrate specificity. Three originally defined nucleotidyltransferase members (EC 2.7.7.16, EC 2.7.7.17, and EC 2.7.7.26) have now been transferred to the hydrolases acting on phosphoric ester bonds (EC 3.1.27). The nucleotidyltransferases involved in chemical reactions include: (a) those involved in polynucleotide synthesis and modification, such as DNA polymerases, RNA polymerases, and CAA-adding enzymes [5, 6]; and (b) those involved in the synthesis, modification and hydrolysis of a wide range of small molecules, such as kanamycin nucleotidyltransferase [7], and GDP–d-glucose phosphorylase. In the Conserved Domain Database, nucleotidyltransferases belong to the Rel-Spo_like superfamily, which includes catalytic domains of Escherichia coli ppGpp synthetase, ppGpp synthetase/hydrolase, and related proteins [8-10]. HEPN domains are widely distributed in many bacteria, archaeons, and vertebrates. In prokaryotes, the HEPN domain has often been found to be associated with the nucleotidyltransferase domain, and is predicted to be involved in nucleotide binding [11, 12].

Bioinformatics studies provide hints regarding some biochemical properties of the Agrobacterium T-complex-recruiting protein Atu5117. It is quite possible that Atu5117 could bind to nucleotides, as the biochemical activities of both putative domains carried by this recruiting protein are related to nucleotides. These conjectures are not enough for us to identify the biochemical function of Atu5117, but it is obvious that knowledge of the biochemical activity of Atu5117 is crucial for understanding how this protein carries out its recruiting function. Therefore, the purpose of this study was to identify the biochemical activity of Atu5117. We demonstrated that Atu5117 was able to hydrolyze eight canonical nucleoside and deoxynucleoside triphosphates with different substrate affinities in vitro, indicating that Atu5117 was a (d)NTPase. The kinetic properties of this (d)NTPase activity were investigated in detail. The characteristics of the (d)NTPase activity imply that Atu5117 might supply energy for the recruitment of T-complex to the transport site.

Results

Overproduction and purification of Atu5117

Atu5117 and its variant with two amino acid substitutions (H218A and R260A) were overproduced by engineered E. coli strains as His-tagged proteins, and purified by affinity chromatography with Ni2+–nitrilotriacetic acid metal affinity resin. In the His-tagged construct, a cleavage site ((Asp)4-Lys-Asp) for enterokinase was introduced between the His tag and the fused protein. Therefore, His tags in the fusion proteins could be removed by enterokinase. To obtain soluble Atu5117 and its variant in amounts sufficient for biochemical characterization, the expression conditions and purification procedure for these proteins were optimized. The purity of these purified proteins was checked by SDS/PAGE. As shown in Fig. 1, four highly pure proteins, His-tagged Atu5117 His-tagged Atu5117 variant, Atu5117 with a five residue (Asp-Pro-Ser-Ser-Arg) N-terminal extension, and Atu5117 variant with a five residue (Asp-Pro-Ser-Ser-Arg) N-terminal extension, were obtained for further biochemical characterization.

Figure 1.

Purification of E. coli-produced Atu5117 and Atu5117 variant. Lane M: molecular mass marker. Lane 1: crude extract of E. coli cells producing His-tagged Atu5117. Lane 2: His-tagged Atu5117 purified on an Ni2+–nitrilotriacetic acid affinity column. Lane 3: Atu5117 with His tag removed. Lane 4: crude extract of E. coli cells producing His-tagged Atu5117 variant. Lane 5: His-tagged Atu5117 variant purified on an Ni2+–nitrilotriacetic acid affinity column. Lane 6: Atu5117 variant with His tag removed.

The apparent molecular mass of native Atu5117 was estimated by size exclusion chromatography. The elution profile of Atu5117 showed a single peak, and the elution volume of Atu5117 was in between those of BSA (66 kDa) and carbonic anhydrase (29 kDa) (Fig. S1). The molecular mass of Atu5117 monomer was calculated to be 36 kDa, suggesting that Atu5117 is a monomer in solution.

Identification of (d)NTPase activity

Bioinformatics analysis showed that Atu5117 contains a putative nucleotidyltransferase domain in its N-terminal region and a putative HEPN domain in its C-terminal region. Initially, our intention was to determine whether the C-terminal putative HEPN domain in Atu5117 could bind any nucleotide. To confirm that any identified biochemical activity was specific to Atu5117, and did not result from any possible contaminating protein or other artefacts from the whole experimental process, an Atu5117 variant with two amino acid substitutions, H218A and R260A, was constructed as a control, because His218 and Arg260 are highly conserved amino acids in the HEPN domain, and, more importantly, mutations in Atu5117 leading to the H218A and R260A substitutions have been shown to abolish the biological function of Atu5117 [2]. As a control, the Atu5117 variant was also produced, purified and subjected to the binding assay in the same manner as native Atu5117.

His-tagged Atu5117 and His-tagged Atu5117 variant were immobilized on Ni2+–nitrilotriacetic acid resin by their His tags. The (d)NTP mixture was incubated with immobilized His-tagged Atu5117 or His-tagged Atu5117 variant. The total UV absorbance in the (d)NTP mixture was monitored during the incubation, but no change in the total UV absorbance in the (d)NTP mixture was observed. Surprisingly, additional chromatogram peaks were found in the chromatogram of the His-tagged Atu5117-incubated (d)NTP mixture when the chemical composition of the (d)NTP mixture was analyzed by reversed-phase ion-pair HPLC (Fig. 2Ad), whereas no additional peak appeared in the chromatogram of the His-tagged Atu5117 variant-incubated (d)NTP mixture (Fig. 2Ab), as compared with the preincubated (d)NTP mixture (Fig. 2Aa,c). On comparison with the retention times of standard (deoxy)nucleotides, these additional chromatogram peaks were identified to be those of (deoxy)nucleoside diphosphates, implying that His-tagged Atu5117 catalyzed the hydrolysis of (d)NTPs. To quantitate the (d)NTPase activity, the peak areas in the chromatogram were calculated to represent the concentration of the corresponding (d)NTP, and the ratio of peak area after incubation to peak area before incubation was defined as the remaining concentration. As shown in Fig. 2B, the remaining concentrations of eight (d)NTPs in the His-tagged Atu5117-incubated (d)NTP mixture were significantly lower than those in the His-tagged Atu5117 variant-incubated (d)NTP mixture, demonstrating that the (d)NTPase activity manifested by the His-tagged Atu5117 was intrinsic to Atu5117, and did not depend on the His tag and the N-terminal five-residue (Asp-Pro-Ser-Ser-Arg) extension or any other contaminating (d)NTPase activity. The data in Fig. 2B also show that His-tagged Atu5117 seemed to prefer NTPs to dNTPs.

Figure 2.

(d)NTPase activiy of Atu5117. His-tagged protein was immobilized on Ni2+–nitrilotriacetic acid affinity resin, and incubated with the nucleotide mixture containing eight canonical (d)NTPs (ATP, CTP, GTP, UTP, dTTP, dATP, dGTP, and dCTP). The concentrations of eight (d)NTPs in the nucleotide mixture before and after incubation with His-tagged protein were analyzed by reversed-phase ion-pair HPLC. (A) Representative chromatograms of the nucleotide mixture before and after incubation with His-tagged proteins. (a) Chromatogram of the nucleotide mixture before incubation with His-tagged Atu5117 variant. (b) Chromatogram of the nucleotide mixture after incubation with His-tagged Atu5117 variant. (c) Chromatogram of the nucleotide mixture before incubation with His-tagged Atu5117. (d) Chromatogram of the nucleotide mixture after incubation with His-tagged Atu5117. (B) Remaining concentrations of eight (d)NTPs in the nucleotide mixture after incubation. Concentrations were calculated from the peak areas. The remaining concentration was defined as the ratio of peak area after incubation to the peak area before incubation. The data represent an average of three independent experiments. Concentrations of eight (d)NTPs in the nucleotide mixture before incubation with His-tagged protein were defined as 1. Filled bar: remaining concentrations of eight (d)NTPs in the nucleotide mixture after incubation with the His-tagged Atu5117 variant. Gray bar: remaining concentrations of eight (d)NTPs in the nucleotide mixture after incubation with His-tagged Atu5117.

Binding of Atu5117 to the ATP analog 3′(2′)-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP)

As Atu5117 was able to hydrolyze (d)NTPs, it should bind to its substrates. We used the fluorescent ATP analog TNP-ATP to verify the binding of Atu5117 to its substrates. The fluorescent TNP-nucleotides have been widely used to characterize the nucleotide-binding abilities of proteins, because the binding of TNP-nucleotides by protein causes a considerable increase in fluorescence, with the absolute magnitude being dependent on the specific protein environment [13-17]. Figure 3 shows that Atu5117 produced significant fluorescence enhancement, whereas the fluorescence of the Atu5117 variant incubated with TNP-ATP remained unchanged. The canonical nucleotide ATP was used to confirm that the binding of Atu5117 to TNP-ATP was specific. The spectra in Fig. 3 show that the addition of ATP to the mixture of Atu5117 and TNP-ATP considerably reduced the fluorescence, suggesting that ATP competitively binds to Atu5117.

Figure 3.

ATP-binding ability of Atu5117. Purified Atu5117 or Atu5117 variant (2.5 μm) was mixed with TNP-ATP (10 μm) in the presence or absence of ATP (1 mm). Fluorescence spectra, from top to bottom, were taken from the following samples: spectrum 1, Atu5117 plus TNP-ATP; spectrum 2, Atu5117 plus TNP-ATP in the presence of ATP; spectrum 3, Atu5117 variant plus TNP-ATP; spectrum 4, TNP-ATP; spectrum 5, Atu5117 variant; spectrum 6, Atu5117. Fluorescence intensities are expressed in arbitrary units (a.u.).

Determination of the optimum conditions for (d)NTPase activity of His-tagged Atu5117

To further characterize the (d)NTPase activity of Atu5117, assessment of the (d)NTPase activity was performed by evaluating the release of inorganic phosphate with a colorimetric assay, as described in 'Experimental procedures'. In the colorimetric assay of (d)NTPase activity, the Atu5117 variant was still used as a control to eliminate any possibility of contaminating proteins and other artefacts. To determine the optimum conditions for nucleotide hydrolysis by His-tagged Atu5117, the effects of pH, temperature and Mg2+ on the (d)NTPase activity of His-tagged Atu5117 were examined. Three substrates, ATP, UTP, and dGTP, were chosen for the determination of the optimum conditions for nucleotide hydrolysis. To ensure a very tight linear relationship between the release of inorganic phosphate and enzymatic reaction time during the reaction period, the enzymatic reaction was carried out with these three substrates at a concentration of 5.0 mm.

In order to determine the optimum pH for the (d)NTP hydrolysis activity of His-tagged Atu5117, the (d)NTPase activities at different pH values were measured in a set of buffers, including 30 mm sodium citrate (pH 4.5, 5.0, and 5.5), 30 mm Mes/NaOH (pH 6.0 and 6.5), 30 mm Tris/HCl (pH 7.0, 7.5, and 8.0), and 30 mm glycine/NaOH (pH 8.5 and 9.0). The optimal pH range for the (d)NTPase activity was observed at pH 5.5–6.5 (Fig. 4A), suggesting that Atu5117 preferred a weak acid medium. Notably, different substrates did not affect the response of (d)NTP hydrolysis activity to pH, although the maximum hydrolysis activity with the substrate ATP at optimal pH was five-fold higher than that with the substrate dGTP. The response of His-tagged Atu5117 activity to temperature was examined over the temperature range of 10–55 °C. The optimum temperature for (d)NTPase activity of His-tagged Atu5117 was in the range 25–32 °C (Fig. 4B). On either side of the optimum temperature, the (d)NTPase activity sharply dropped. The (d)NTPase activity of His-tagged Atu5117 at 55 °C remained ~ 10% of the activity at optimum temperature, implying that His-tagged Atu5117 is unable to tolerate high temperature. Both the optimum pH and temperature for (d)NTPase activity of His-tagged Atu5117 were consistent with the inducing pH and temperature for A. tumefaciens tumorigenesis [18, 19]. Therefore, all of the following (d)NTP hydrolysis reactions by Atu5117 were carried out at 28 °C in 30 mm Mes/NaOH (pH 6.0) buffer solution.

Figure 4.

Effects of pH, temperature and Mg2+ on the (d)NTPase activity of His-tagged Atu5117. (A) Effects of pH on the (d)NTPase activity. (d)NTP hydrolysis was carried out at 28 °C in the buffer indicated in the text, containing 0.5 μm protein, 50 mm NaCl, 6 mm MgCl2, and 5.0 mm substrate. (B) Effects of temperature on the (d)NTPase activity. (d)NTP hydrolysis was carried out at different temperatures in 30 mm Mes/NaOH (pH 6.0) containing 0.5 μm protein, 50 mm NaCl, 6 mm MgCl2, and 5.0 mm substrate; ♦, ATP; ●, UTP; ▲, dGTP. (C) Effects of Mg2+ on the (d)NTPase activity. (d)NTP hydrolysis was carried out at 28 °C in 30 mm Mes/NaOH (pH 6.0) containing 0.5 μm protein, 50 mm NaCl, 5.0 mm substrate, and various concentrations of MgCl2, in the presence or absence of EDTA (2.5 mm). Filled bar: ATP. Gray bar: UTP. Open bar: dGTP. The data are means of three independent measurements with standard deviations.

Most nucleotide phosphohydrolases require Mg2+ as a cofactor to function [20-24]. To examine whether Mg2+ is required for the (d)NTPase activity of His-tagged Atu5117, EDTA was used to chelate Mg2+ that was added in the protein purification. We also tested the effect of Mg2+ on the (d)NTPase activity of His-tagged Atu5117 within an Mg2+ concentration range of 0.5–7.5 mm. The results in Fig. 4C show that Mg2+ was essential for the (d)NTPase activity of His-tagged Atu5117, and that the concentration of MgCl2 affected this activity.

Kinetic analysis and substrate specificity of Atu5117 (d)NTPase

As shown in Fig. 2, His-tagged Atu5117 was able to hydrolyze all eight canonical (d)NTPs. To further confirm the substrate preference of Atu5117, the kinetic parameters of Atu5117 for eight (d)NTPs were measured. Because the additional His tag might affect the catalytic properties of Atu5117, the N-terminal His tag was removed from His-tagged Atu5117 by enterokinase, and only five additional amino acids (Asp-Pro-Ser-Ser-Arg) were kept on the N-terminus of Atu5117. The kinetic parameters of (d)NTPase for different nucleotide substrates were determined by analyzing the effects of substrate concentrations on hydrolysis rates. For assay of the kinetic parameters, the nucleotide substrate concentration was varied from 0.02 to 3.5 mm. The datasets of initial velocity rate and substrate concentration were fitted to the Michaelis–Menten equation (Fig. 5). The kinetic parameters of His-tagged Atu5117 and Atu5117 for eight substrates are summarized in Table 1. The results showed that the catalytic efficiencies of Atu5117 for all eight substrates were significantly higher than those of His-tagged Atu5117; in particular, the kcat values of Atu5117 for four NTPs were approximately two times of those of His-tagged Atu5117, implying that the His tag affected the catalytic activities of Atu5117. However, the influences of the His tag on the affinities of Atu5117 for eight substrates were very different from the influences of the His tag on the activities. The Km values of Atu5117 for four NTPs were much lower than those of His-tagged Atu5117 for four NTPs. For example, the Km value of Atu5117 for ATP was four times lower than that of His-tagged Atu5117. In contrast, the Km values of Atu5117 for four dNTPs were significantly higher than those of His-tagged Atu5117, indicating that removal of the His tag enhanced the selectivity of Atu5117 for four NTPs. We compared the kinetic parameters of Atu5117 (d)NTPase with those of other (d)NTPases [25-29]. The kinetic parameters of Atu5117 (d)NTPase were approximately the same order of magnitude as those of other (d)NTPases. Given that the NTP pool level in bacterial cells has been estimated to be in the order of magnitude of 102–103 μm [30], and physiological concentrations of dNTP might be one order of magnitude lower than those of NTP [31], dNTPs were unable to compete with NTPs for the active site of Atu5117 in vivo, because the Km values of Atu5117 for four dNTPs were not in the order of magnitude of the physiological concentrations of dNTP. Therefore, Atu5117 should be an NTPase, not a dNTPase, in vivo, although it could hydrolyze dNTPs in the in vitro assays.

Table 1. Kinetic parameters of His-tagged Atu5117 and Atu5117 for eight substrates. The hydrolysis reaction was carried out at 28 °C in 30 mm Mes/NaOH (pH 6.0) containing 0.5 μm protein, 50 mm NaCl, 6 mm MgCl2, and different concentrations of substrate. The kinetic parameters were determined in triplicate assays
SubstrateHis-tagged Atu5117Atu5117
Km (mm)kcat (min−1)kcat/Km (mm−1·min−1)Km (mm)kcat (min−1)kcat/Km (mm−1·min−1)
ATP0.57 ± 0.041.92 ± 0.093.370.14 ± 0.013.87 ± 0.2327.64
UTP0.55 ± 0.051.44 ± 0.112.620.18 ± 0.013.01 ± 0.2116.72
GTP0.66 ± 0.061.13 ± 0.091.710.24 ± 0.032.66 ± 0.2211.08
CTP0.61 ± 0.070.96 ± 0.081.570.26 ± 0.021.85 ± 0.127.12
dATP1.12 ± 0.110.58 ± 0.060.521.63 ± 0.110.81 ± 0.070.50
dTTP0.93 ± 0.080.54 ± 0.050.581.78 ± 0.150.57 ± 0.060.32
dGTP1.04 ± 0.110.48 ± 0.040.461.86 ± 0.190.51 ± 0.040.27
dCTP0.98 ± 0.090.43 ± 0.050.441.73 ± 0.120.47 ± 0.050.27
Figure 5.

Kinetic analysis of His-tagged Atu5117 and Atu5117 (d)NTPase activity. The (d)NTP hydrolysis activities were monitored as a function of substrate concentration, and fitted well with a Michaelis–Menten curve (represented by lines). (A) Hydrolysis activity of His-tagged Atu5117 for four canonical NTPs. (B) Hydrolysis activity of His-tagged Atu5117 for four canonical dNTPs. (C) Hydrolysis activity of Atu5117 for four canonical NTPs. (D) Hydrolysis activity of Atu5117 for four canonical dNTPs. The initial velocity rate (y-axis) is expressed as moles of phosphate released per minute per mole of protein (or apparent kinetic rate constant; min−1). The data are means of three independent measurements with standard deviation. ▲, ATP; ◆, UTP; ●, GTP; ■, CTP; △, dATP; ◇, dTTP; ○, dGTP; □, dCTP.

Discussion

Atu5117 is able to recruit T-complex from the cytosol to the T4SS transport site, and is designated as a T-complex recruiting protein [2], but how Atu5117 recruits T-complex is unknown. Sequence analysis indicated that Atu5117 contained a putative nucleotidyltransferase domain and a putative HEPN domain [1], and belonged to a highly conserved protein family. Until now, no protein with a sequence or structure domain that is highly similar to Atu5117 has been identified as a (d)NTPase, although three originally defined nucleotidyltransferase members have now been transferred to hydrolases that act on phosphoric ester bonds. Here, our data demonstrate that Atu5117 is able to hydrolyze four canonical NTPs and four canonical dNTPs in in vitro assays, whereas an Atu5117 variant that was proved to be a function-deficient variant in a previous study [2] was purified exactly in the same way as Atu5117, but was devoid of in vitro (d)NTPase activity, confirming that the (d)NTPase activity of Atu5117 cannot result from a contaminant present in the purified protein preparation. Moreover, the TNP-ATP binding assay provided direct evidence that ATP could bind to Atu5117. All of these data firmly support the idea that Atu5117 is a newly identified (d)NTPase. This novel finding should provide new insights for the characterization of this highly conserved protein family.

Characterization of the (d)NTPase activity of Atu5117 revealed that the optimum pH and temperature for the (d)NTPase activity of Atu5117 accorded with the inducing pH and temperature for tumorigenesis, implying that the (d)NTPase activity of Atu5117 was able to play a role in fulfilling the in vivo biological function of Atu5117. The kinetic parameters of Atu5117 for eight substrates were very different from those of His-tagged Atu5117, demonstrating that the additional His tag significantly influenced the biochemical activities of Atu5117. Comparison of the Km values of Atu5117 for eight substrates with those of other (d)NTPases and physiological concentrations of nucleotides indicated that Atu5117 was an NTPase, not a (d)NTPase, in vivo. The highest specificity and catalytic efficiency of Atu5117 NTPase activity for ATP imply that Atu5117 is a potential candidate as a key ATPase in providing energy for T-complex recruitment. The kcat and Km values of Atu5117 NTPase for ATP are also comparable with those of the ATPases identified in some components of the VirB/D4 T4SS [16, 32-36], indicating that Atu5117 NTPase could use ATP at the same concentration as used by T4SS ATPases. However, the size of the nucleoside pool in bacterial cells is dependent on the physiological state of the cell [30]. As a soilborne plant pathogen, A. tumefaciens often survives under extreme environmental conditions [3, 37]. Atu5117 is able to hydrolyze all four canonical NTPs with high affinity and catalytic efficiency, demonstrating that it could use broad energy sources that are not limited to ATP for the recruitment of T-complex to the transport site.

In conclusion, our study is the first rigorous demonstration of a (d)NTPase activity for a protein with putative nucleotidyltransferase and HEPN domains. As Atu5117 is a T-complex-recruiting protein, this (d)NTPase activity might energize T-complex recruitment. These findings are important not only for our understanding of the mechanism of T-complex recruitment by Atu5117, but also for the characterization of the biochemical function and property of proteins containing nucleotidyltransferase and HEPN domains.

Experimental procedures

Plasmid construction

The gene for Atu5117 was amplified by PCR with A. tumefaciens C58 genomic DNA as template. The primer sequences for this PCR were 5′-CCCTCGAGGAAAACATCGCTCGATCATATT-3′ and 5′-AAAAAGCTTTAGCCCGCTATTCTTCAG-3′ (forward and reverse, respectively), where the underlined nucleotides indicate the restriction sites of XhoI (CTCGAG) and HindIII (AAGCTT). The resulting PCR product was digested with XhoI and HindIII, and inserted into the same sites of the pRSET B expression vector to express His-tagged Atu5117. The Atu5117 mutant with two mutation sites (H218A and R260A) was constructed by using PCR according to Guo et al. [2, 38]. The Atu5117 mutant was also expressed by the pRSET B expression vector as a His-tagged fusion protein. Escherichia coli DH5α strain was used as a host for the gene clone.

Overproduction and purification of protein

E. coli BL21(DE3) was used as a host to overproduce His-tagged Atu5117 and His-tagged Atu5117 variant. E. coli BL21 harboring one of the recombinant pRSET B plasmids for wild-type Atu5117 or mutant Atu5117 was grown to approximately 5 × 108 cells·mL−1, and isopropyl thio-β-d-galactoside was then added to the cultures at a final concentration of 0.3 mm to induce fusion protein expression. After additional cultivation at 37 °C for 2–3 h, the cells in the culture were collected by centrifugation at 5000 g for 5 min, and resuspended in lysis buffer (100 mm NaCl, 2 mm phenylmethanesulfonyl fluoride, 20 mg·L−1 leupeptines, 50 mm Tris/HCl, pH 7.4). All of the following procedures were carried out at 4 °C. The cell suspension was sonicated to near clarity, and centrifuged at 18 000 g for 20 min. The supernatant was incubated with pre-equilibrated Ni2+–nitrilotriacetic acid metal affinity resin (Qiagen, Hilden, Germany) at 4 °C for 1 h. After His-tagged Atu5117 (or its variant) was bound to the Ni2+–nitrilotriacetic acid metal affinity resin, the resin was loaded onto a column and washed with lysis buffer containing 30 mm imidazole. His-tagged Atu5117 (or its variant) was eluted with elution buffer (100 mm NaCl, 150 mm imidazole, 50 mm Tris/HCl, pH 7.4). The eluted His-tagged protein was dialyzed against binding buffer (50 mm NaCl, 2.5 mm MgCl2, 30 mm Tris/HCl, pH 7.4) and concentrated. The His tag in the fusion protein was removed by use of the Enterokinase Cleavage Capture Kit (Novagen, EMD Biosciences, Madison, WI, USA), leaving only a five-residue (Asp-Pro-Ser-Ser-Arg) N-terminal extension in Atu5117. For the removal of His tag, the His-tagged protein-bound Ni2+–nitrilotriacetic acid resin was washed with EK buffer (50 mm NaCl, 20 mm Tris/HCl, 2 mm CaCl2, pH 7.4), and then incubated with recombinant enterokinase at room temperature overnight. Atu5117 or its variant could be cleaved from the His-tagged protein-bound Ni2+–nitrilotriacetic acid resin. The recombinant enterokinase in the protein solution was removed with EKapture Agarose, according to the Novagen technical manual. Protein purity in the purification process was monitored by SDS/PAGE. The protein concentration was determined by the Bradford method, with BSA as a standard.

Determination of apparent molecular mass

Size exclusion chromatography was used to determine the apparent molecular mass of Atu5117. The protein sample (100 μg in 100 μL) was loaded onto a pre-equilibrated Sephadex G-100 column, and eluted with 30 mm Tris/HCl (pH 7.4) buffer containing 50 mm NaCl and 2.5 mm MgCl2 at a flow rate of 0.5 mL·min−1. The eluted protein was monitored with a UV detector. BSA (66 kDa) and carbonic anhydrase (29 kDa) were used as standard molecular mass markers. The apparent molecular mass of Atu5117 was verified by comparing its elution volume with those of two markers.

Nucleotide-binding assay and identification of nucleotide hydrolysis

Nucleotide-binding assay was carried out by incubating the His-tagged protein-bound Ni2+–nitrilotriacetic acid resin with a nucleotide mixture that contained eight canonical nucleoside and deoxynucleoside triphosphates (ATP, CTP, GTP, UTP, dATP, dCTP, dGTP, and dTTP), with a 2.5 μm final concentration of each nucleotide in binding buffer. For the nucleotide-binding assay, 3 nmol of purified His-tagged Atu5117 was rebound to 500 μL of slurry (50% v/v) of Ni2+–nitrilotriacetic acid resin, so that the concentration of His-tagged Atu5117 could be maintained at 6 μm in the binding assay. The fusion protein-bound Ni2+–nitrilotriacetic acid resin was washed with reaction buffer and incubated with the nucleotide mixture at 20 °C for 5 min. The purified His-tagged Atu5117 variant was used as a negative control, and an equal amount of bare Ni2+–nitrilotriacetic acid resin was used as a blank control in the assay. The total concentration of nucleotides in the nucleotide mixture was determined from the absorbance at 260 nm, with a UV–visible spectrophotometer. The binding of nucleotides to the His-tagged Atu5117 was monitored by the difference in UV absorbance of the nucleotide mixture after and before incubation with the His-tagged protein-bound Ni2+–nitrilotriacetic acid resin. To identify nucleotide hydrolysis, nucleotides in the nucleotide mixture after incubation with the His-tagged protein-bound Ni2+–nitrilotriacetic acid resin were separated by reversed-phase ion-pair HPLC, according to Pierro et al. [39] and Williams et al. [40]. Chromatographic separation was performed with an Agilent1200 LC system equipped with a ZORBAX Eclipse XDB C18 column (Agilent Technologies, Santa Clara, CA, USA). The column temperature was maintained at 27 °C. The flow rate of the mobile phase was 1.0 mL·min−1. The wavelength of the UV detector was set at 254 nm. The injection volume was 20 μL. The elution buffer was prepared and the elution program was designed as described by Huang et al. [41].

Alternatively, the fluorescent ATP nucleotide analog TNP-ATP (Molecular Probes, Eugene, OR, USA) was used to further investigate nucleotide binding. Protein solution and TNP-ATP solution were prepared at 2.5 μm and 10 μm, respectively, in measuring buffer (30 mm Tris/HCl, pH 7.4, 50 mm NaCl). The mixture of protein and TNP-ATP was incubated at room temperature for 1 min in the presence or absence of 1 mm ATP. Fluorescence spectra were taken at room temperature with excitation at 410 nm, and emission was scanned in the range 470–630 nm. All spectra were corrected by subtracting the background fluorescence emitted from measuring buffer solution.

(d)NTPase assay

(d)NTPase activity was measured by monitoring the amount of inorganic phosphate released upon nucleotide hydrolysis. Inorganic phosphate was determined with the malachite green–ammonium molybdate assay [13, 20, 42], with minor modifications. In the standard assay of (d)NTPase activity, the nucleotide hydrolysis reaction was carried out at 28 °C in 30 mm Mes/NaOH (pH 6.0) buffer containing 0.5 μm protein (His-tagged Atu5117, Atu5117, His-tagged Atu5117 variant, or Atu5117 variant), 50 mm NaCl, 6 mm MgCl2, and the indicated concentration of nucleotide substrate. Samples were taken from the reaction mixture at different time points, and were mixed with the color reagent. The absorbance of the color complex was measured at 630 nm. Each sample was assayed in triplicate. The amount of inorganic phosphate released was calculated from a standard curve obtained with NaH2PO4. The time course of inorganic phosphate release was used to confirm that steady-state conditions were established, and to measure the initial velocity. All readings were corrected by subtracting the background values from the indicated controls.

Acknowledgements

We thank H. Xie for technical assistance with HPLC. This work was funded jointly by the National Science Foundation of China (30870054, 31170073) and the Funding Plan for the High-level Talents with Overseas Education to Work in China from the Ministry of Human Resources and Social Security of China.

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