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J. Biol. Chem., Vol. 282, Issue 19, 14018-14027, May 11, 2007

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ATP Binding to the KTN/RCK Subunit KtrA from the K⁺-uptake System KtrAB of Vibrio alginolyticus

ITS ROLE IN THE FORMATION OF THE KtrAB COMPLEX AND ITS REQUIREMENT IN VIVO^*

From the Department of Microbiology, University of Osnabrück, D-49076 Osnabrück, Germany

Received for publication, September 25, 2006 , and in revised form, February 20, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Subunit KtrA of the bacterial Na⁺-dependent K⁺-translocating KtrAB systems belongs to the KTN/RCK family of regulatory proteins and protein domains. They are located at the cytoplasmic side of the cell membrane. By binding ligands they regulate the activity of a number of K⁺ transporters and K⁺ channels. To investigate the function of KtrA from the bacterium Vibrio alginolyticus (VaKtrA), the protein was overproduced in His-tagged form (His¹⁰-VaKtrA) and isolated by affinity chromatography. VaKtrA contains a G-rich, ADP-moiety binding ---fold ("Rossman fold"). Photocross-linking and flow dialysis were used to determine the binding of [³²P]ATP and [³²P]NAD⁺ to His¹⁰-VaKtrA. Binding of other nucleotides was estimated from the competition by these compounds of the binding of the ³²P-labeled nucleotides to the protein. [-³²P]ATP bound with high affinity to His¹⁰-VaKtrA (K_D of 9 µM). All other nucleotides tested exhibited K_D (K_i) values of 30 µM or higher. Limited proteolysis with trypsin showed that ATP was the only nucleotide that changed the conformation of VaKtrA. ATP specifically promoted complex formation of VaKtrA with the His-tagged form of its K⁺-translocating partner, VaKtrB-His⁶, as detected both in an overlay experiment and in an experiment in which VaKtrA was added to VaKtrB-His⁶ bound to Ni²⁺-agarose. In intact cells of Escherichia coli both a high of membrane potential and a high cytoplasmic ATP concentration were required for VaKtrAB activity. C-terminal deletions in VaKtrA showed that for in vivo activity at least 169 N-terminal amino acid residues of its total of 220 are required and that its 40 C-terminal residues are dispensable.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The major solute in the cytoplasm of all cells, K⁺, plays an important role in osmo-regulation. Many bacteria react to hyperosmotic shock by rapidly accumulating K⁺ from the medium, thereby restoring their turgor pressure (1–3). For this function, bacteria possess at least three types of K⁺-uptake systems, Kdp, Trk, and Ktr (2, 4, 5). These systems are all composed of several types of subunits and mediate a tightly controlled mode of K⁺ transport. The mechanism by which they accomplish this differs from system to system. The activities of the K⁺-translocating P-type ATPase Kdp and of the K⁺-transporter Trk are directly influenced by the magnitude of the cell turgor pressure (6). Trk consists of three types of subunits: TrkE/SapD that binds ATP (7, 8), TrkA that binds NAD(H) (9), and TrkG/TrkH which translocates K⁺ across the membrane (2). The third regulated bacterial K⁺-uptake system is Ktr (4, 10, 11). Its activity depends on Na⁺ ions (12). With only two types of subunits, the KtrAB system from Vibrio alginolyticus (VaKtrAB)⁴ has a relatively simple composition. The K⁺-translocating subunit KtrB is a member of the SKT proteins, which are believed to have evolved from simple K⁺ channels by multiple gene duplications and gene fusions (13, 14). KtrA is the regulatory subunit. It is a membrane surface protein. KtrA confers velocity, ion specificity, and ion coupling to the Ktr system (15). Structural work on a NAD⁺ binding N-terminal KtrA fragment from Bacillus subtilis compared with that of an NADH binding N-terminal TrkA fragment from Methanocaldococcus jannaschii has led Roosild et al. (16) to propose that KtrB activity is activated by a conformational switch brought about by the replacement of NAD⁺ by NADH as the ligand bound to KtrA. However, recent data show that ATP rather than nicotinamide nucleotides binds with the highest affinity to the KtrA fragment (K_D about 600 nM (17)). KtrA belongs to the family of KTN proteins, which are closely related to cytoplasmic RCK domains of several types of K⁺ channels (Refs. 18–21; see supplemental Fig. S1 for an alignment of VaKtrA with that of KTN/RCK domains of known structure). The N-terminal part of these proteins (i.e. _A to _E; supplemental Fig. S1) possesses a Rossmann-fold type of - protein structure, closely similar to that of the NAD⁺ binding domains of NAD⁺-dependent dehydrogenases (2, 9, 22–24). In the N-terminal - domain, the glycine-rich sequence (GXGXXG...(D/E)) is important for the binding of the adenosine moiety of NAD(H). However, in several RCK domains glycine residues from this fold are replaced by other amino acids (supplemental Fig. S1). The K⁺ channel Mth from Methanobacterium thermoautotrophicum is one of these proteins. It binds Ca²⁺, and its activity depends on the presence of this divalent cation (19, 25). For a second K⁺ channel, Kch from Escherichia coli, it is not known which ligand binds to its RCK domain (18).

Because of these uncertainties about ligand binding to KTN/RCK domains and their role in the regulation of both K⁺ transport and K⁺ channel activity, we examined nucleotide binding to the isolated full-length KtrA protein from the bacterium V. alginolyticus. We show that ATP binds with higher affinity than does NAD⁺ or NADH, that ATP promotes complex formation between KtrA and KtrB, and that in vivo VaKtrAB-expressing E. coli cells require ATP and the membrane potential for activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Strains, Plasmids, Growth Conditions—The strains and plasmids used in this study are listed in supplemental Table S1. Plasmid pEL305 contains VaKtrA cloned into the NdeI and BamHI sites of plasmid pET16b (Novagen, Schwalbach, Ts, Germany). It encodes KtrA with the 21-amino acid N-terminal extension MGHHHHHHHHHHSSGHIEGRH (His¹⁰-KtrA). Plasmid pKT84 (4) contains VaKtrAB cloned into vector pHG165 (26). Plasmids pMW130stop to pMW180stop were generated by PCR using the QuikChange site-directed mutagenesis kit from Stratagene, La Jolla, CA. They contain a stop codon at positions 130 to 180 of KtrA in plasmid pKT84, respectively. Plasmid pEL903 encodes VaKtrB-His⁶ under the control of the p_ara promoter of plasmid pBAD18 (27). Plasmid-containing cells of strains LB2003 and LB692 were grown at 37 °C under aerobic conditions in minimal medium K30 (28) containing in addition 1 mg/liter thiamine, 40 mg/liter methionine (only strain LB2003), 100 mg/liter carbenicillin, and 10 mM glucose. For overproduction of His¹⁰-KtrA, plasmid pEL305-containing cells of strain BL21(DE3)pLysS (29) were grown at 30 °C under aerobic conditions in KML medium containing 10 g/liter KCl, 10 g/liter Tryptone, 5 g/liter yeast extract, 100 mg/liter ampicillin, and 34 mg/liter chloramphenicol. For production of KtrB-His⁶, cells of strain Rosetta^TM2(DE3)/pEL903 were grown as described in Tholema et al. (15).

Complementation of K⁺ Transport by Growth Measurements; Net K⁺-uptake Activity—These assays were done as described previously (12, 15, 28).

Detection of C-terminal-truncated KtrA in a Minicell System—Plasmid pMWstop-encoded, C-terminal-truncated KtrA proteins labeled with [³⁵S]methionine were detected in minicells of E. coli DK6 (30) as described in Nakamura et al. (31).

Experiments on Energy Coupling of VaKtr—Cells of strain LB692/pKT84 were depleted of ATP and K⁺ by incubation with 2,4-dinitrophenol as described in Harms et al. (8). For the transport assays cells were preincubated for 30 min at 20 °C with 10 mM glucose, 20 mM disodium succinate or without substrate. Transport was initiated by the addition at t = 0 of 1 mM KCl, 2 µM [¹⁴C]glutamine (40 nCi/ml), or 4 µM [¹⁴C]proline (100 nCi/ml) to the cell suspension. If present, 2,4-dinitrophenyl was added at t =–5 min. Uptake of K⁺ or radioactive compounds by the cells was measured as a function of time, as described in Harms et al. (8). The ATP content of these cells was determined by the method of Kimmich et al. (32).

Overproduction and Purification of His¹⁰-KtrA—Overnight grown cells of E. coli BL21 (DE3)pLysS/pEL305 were transferred at an OD₅₇₈ value of about 0.05 to 4 1-liter portions of fresh KML medium and grown at 30 °C. 1 mM isopropyl 1-thio--D-galactopyranoside was added at OD₅₇₈ of 0.25–0.3. After a further 20 min the cultures received 0.1 g/liter rifampicin. The cultures were shaken for an additional 2 h. Subsequently the cells were harvested by centrifugation at 10,000 x g. The cell pellets from 1 liter of cell culture were suspended in 18 ml of buffer A, subsequently frozen with liquid nitrogen, and stored at –80 °C. Buffer A contained 300 mM NaCl, 1 mM EDTA, and 50 mM Tris-HCl, pH 8.0.

For cell fractionation 18 ml of cell suspension in buffer A plus 180 µl of protease inhibitor mixture P8849 (Sigma/Aldrich) were sonicated 5 times for 30 s each (50% duty cycle) with maximal output from the large tip of a Branson Sonifer (Branson, Heusenstamm, Germany). Cell debris and the membrane fraction were removed by subsequent centrifugation steps at 28,000 x g for 15 min and 350,000 x g for 30 min, respectively. The supernatant of the latter (cell fraction of soluble proteins) was stored in liquid nitrogen.

For affinity purification of recombinant His¹⁰-VaKtrA, prepacked protino®Ni 2000 column (Machery-Nagel GmbH & Co. KG, Düren Germany) preincubated with the supplied buffer plus 5 mM imidazole, 200 mM NaCl, and 30 mM 2-mercaptoethanol was used. After His¹⁰-VaKtrA binding at 5 mM imidazole, the column was washed with 50 ml of buffer containing 5 mM imidazole. His¹⁰-VaKtrA was eluted from the column in four steps by the successive addition of 800 µl of buffer containing 250 mM imidazole to the column. Imidazole was removed from the purified His¹⁰-VaKtrA fractions by buffer exchange against 0.2 M NaCl, 20 mM NaH₂PO₄, 2 mM 2-mercaptoethanol, 1 mM EDTA, pH 7.0 (buffer B) on NAP-5 columns (Amersham Biosciences). Purified His¹⁰-VaKtrA was stored at 4 °C, at which it remained in solution for several days.

Removal of the His Tag from His¹⁰-VaKtrA—The tag was removed by digestion for 16 h at 20 °C with 50 units of protease Factor Xa (Novagen)/mg in buffer B containing 3–5 mg/ml His¹⁰-VaKtrA protein. Subsequently, the split-off His tag was removed on a NAP-5 column. The product (VaKtrA) contains a histidine residue as the N-terminal extension.

Trypsin Digestion of His¹⁰-VaKtrA—About 60 µg of His¹⁰-VaKtrA in 300 µl of buffer B containing either no nucleotides, 1 mM NAD⁺, 1 mM NADH, or 1 mM ATP were incubated with 1.2 µg of trypsin. 50-µl samples were removed at different times and mixed immediately with an equivalent amount of soybean trypsin inhibitor. The protein pattern of the samples was analyzed by SDS-PAGE. Some fragments were identified by N-terminal sequencing.

Antibodies—Rabbit polyclonal antibodies against purified VaKtrA were produced by Charles Rivers, Kisslegg, Germany. Monoclonal anti-penta-His antibody was purchased from Qiagen, Hilden, Germany.

Overlay Experiment—VaKtrB-His⁶-containing membranes from strain Rosetta2(DE3)/pEL903 were prepared as described in Tholema et al. (15). Proteins from this fraction were separated by SDSPAGE and transferred to a nitrocellulose sheet where they were stained with Ponceau Red. Separate protein lanes were cut out from the sheet. After removal of the stain and treatment with 3% of the blocking agent bovine serum albumin, these lanes were incubated with purified VaKtrA in the absence or presence of nucleotides at the concentration specified in the legend to Fig. 5. KtrA was detected with its specific antibody used at a 1:300,000 dilution followed by horseradish peroxidase-conjugated secondary antibodies (1:1000 dilution) and visualization with 4-nitro-blue-tetrazolium chloride/5-bromo4-chloro-3-indolylphosphate according to the recommendations of the supplier (Roche Diagnostics). In parallel, VaKtrB-His⁶ in the protein lane was detected with the monoclonal anti-penta-His antibody.

Binding of Solubilized VaKtrB-His₆ to Ni²⁺-agarose—VaKtrB-His⁶ was solubilized from membranes of strain Rosetta2(DE3)/pEL903 as described in Tholema et al. (15), except that membranes were present at 20 mg/ml and that the detergent n-dodecyl -D-maltoside was present at 1.3% (w/v). Solubilized His-tagged protein was bound to Ni²⁺-agarose (Qiagen) in the presence of 5 mM imidazole. Accompanying, non-His-tagged proteins were eluted by washing the column with 50 and 8 bed volumes of buffer containing 30 and 50 mM imidazole, respectively.

Complex Formation between VaKtrA and VaKtrB-His⁶ Bound to Ni²⁺-agarose; Elution of the Complex—An amount of 1 ml of column material containing about 0.2 mg of bound VaKtrB-His⁶ was incubated with 0.5 ml of buffer B without EDTA containing 0.5 mg of VaKtrA and either 100 µM ATP or no nucleotide. Subsequently the column was washed again with 50 and 8 bed volumes of buffer containing 30 and 50 mM imidazole, respectively. The KtrAB complex was eluted with buffer containing 250 mM imidazole. Detection of KtrA or KtrB-His⁶ was done with the specific antibodies as described above for the overlay experiment.

Photochemical Binding of Adenine-(di)-nucleotides—Photochemical binding of [³²P]NAD⁺ or [-³²P]ATP (Amersham Biosciences; Ref. 33) to His¹⁰-VaKtrA was done as in Schlösser et al. (9). Samples of 20 µl of buffer B containing 10–40 µg of protein, 0.5–1 µCi of ³²P-labeled compound (specific activity 500–1000 Ci/mmol), and varying concentrations of nonradioactive (di)-nucleotides were irradiated with ultraviolet light for 20–40 min on ice. Subsequently each sample received 20 µl of 2x-concentrated sample solubilization buffer (8% SDS, 24% glycerol, 100 mM Tris-HCl, 4% 2-mercaptoethanol, 0.02% Serva blue, pH 6.8), and the mixture was incubated for 30 min at 40 °C. Proteins from 20-µl samples were separated by SDS-PAGE. Radioactivity on the dried gel was detected and quantified by phosphorimaging using an Amersham Biosciences Storm 820 apparatus.

Flow Dialysis for Determining Nucleotide Binding to His¹⁰-VaKtrA—Binding of nucleotides to His¹⁰-VaKtrA was determined at room temperature by flow dialysis (34). The upper chamber was filled with 1 ml of buffer D containing 2 mM -mercaptoethanol, 0.2 M NaCl, 1 mM EDTA, 20 mM sodium phosphate, pH 7.0, and radioactivity (1 µCi (about 1–5 pmol) of [³²P]NAD⁺ or [-³²P]ATP) in the absence or presence of 1–2 mg of His¹⁰-VaKtrA. The lower spiral chamber was separated from the upper chamber by a Visking type 36 dialysis membrane (Biomol, Hamburg, Germany). Buffer D was passed through the lower chamber at a rate of about 1 ml/min. 0.8-ml samples of the dialysate were collected and mixed with 4 ml of scintillation fluid Ecolume (ICN, Heidelberg, Germany), and their radioactivity was determined in a Tricarb 2300 TR liquid scintillation counter (PerkinElmer Life Sciences). Binding of radioactivity to His¹⁰-VaKtrA was calculated from the difference between radioactivity in the dialysate in the absence and presence of His¹⁰-VaKtrA. The K_D-value for ATP binding and the amount of ATP maximally bound to His¹⁰-KtrA on a molar basis were determined according to the method of Scatchard (35). For this calculation the molar concentration of His¹⁰-VaKtrA was determined spectroscopically using a ₂₈₀ value of 5,600 M^–1·cm^–1, calculated from the contributions at that wavelength of the one tryptophan and five tyrosine residues per VaKtrA molecule (4). Binding constants for nucleotides other than ATP were determined by first binding a small amount of radioactive ATP to His¹⁰-VaKtrA and then gradually releasing the radioactivity by adding increasing concentrations of the second nucleotide. For this calculation it was assumed that the K_D value for ATP was 9 µM and that KtrA possesses a single nucleotide binding site.

View larger version (30K): [in this window] [in a new window]   FIGURE 1. Isolation of His10-VaKtrA by affinity chromatography (A) and the removal of its His tag (B). His10-VaKtrA was produced in E. coli BL23(DE3)pLysS/pEL305 and was isolated from the fraction of soluble proteins as described under "Experimental Procedures." Panel A gives the fraction of soluble proteins (lane 1), the flow-through fraction of soluble proteins with the Ni2+-agarose column (lane 2), wash fractions with 5 mM imidazole (lanes 3 and 4), His10-VaKtrA elution fractions with 250 mM imidazole (lanes 5–9), respectively. Panel B, isolated His10-VaKtrA before and after overnight digestion with factor Xa protease.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Overproduction and Purification—VaktrA was cloned into the NdeI and BamHI sites of the expression plasmid pET16b, giving plasmid pEL305. This plasmid encodes VaKtrA with a 21-residue N-terminal extension of 10 histidine residues followed by a factor Xa proteolytic cleavage site (His¹⁰-VaKtrA). Overproduction of His¹⁰-VaKtrA in strain BL21(DE3)pLysS/pEL305 gave good yields of the protein in both the cell membrane and soluble protein fractions. From the latter, His¹⁰-VaKtrA was isolated by affinity chromatography on nickel-agarose (Fig. 1A). The yield was about 250 mg of His¹⁰-VaKtrA/l cell culture, and the preparation had a purity of at least 95% (Fig. 1A, elution fractions 1–4), which was sufficient for the intended experiments. His¹⁰-VaKtrA ran as a single peak with an apparent M_r of about 280,000 on a Sepharose 200 column (data not shown), suggesting that His¹⁰-VaKtrA is a decamer. Because this type of M_r determination is inaccurate, our data are compatible with KtrA being an octamer (17).

Photochemical Binding of [³²P]NAD⁺ to His¹⁰-VaKtrA—Because the first two KTN protein fragments for which a structure was known were complexed with NAD⁺ and NADH, respectively (16), we started our nucleotide binding studies with [³²P]NAD⁺. Previously we have shown that [³²P]NAD⁺ can be photochemically bound to the KTN protein TrkA from E. coli (9). Hence we applied the same technique to His¹⁰-VaKtrA (Fig. 2). After strong illumination of His¹⁰-VaKtrA with light of 260 nm in the presence of [³²P]NAD⁺, the protein became radioactively labeled. This process was prevented by increasing concentrations of nonradioactive NAD⁺ (Fig. 2A, lanes 2–4). Quantification of the amount of radioactive NAD⁺ bound to His¹⁰-VaKtrA showed that under the assay conditions of Fig. 2, only a few percent of VaKtrA had bound the radioactive nucleotide.

View larger version (76K): [in this window] [in a new window]   FIGURE 2. Photochemical binding of [32P]NAD+ and [-32P]ATP to His10-VaKtrA. Panels A–C, labeling with [32P]NAD+, except for B, lanes 9–12, where labeling was with [-32P]ATP. The concentrations of inhibitory nonradioactive nucleotides was as shown in the figure. The far left lane in panel A is a Coomassie-stained preparation of purified His10-VaKtrA. Panel D, competition of nonradioactive nucleotides with the photocross-linking of [32P]NAD+ or [-32P]ATP with His10-VaKtrA. The intensities of the bands in panels A and B were quantified by phosphorimaging and plotted as a percent of the control values (no nonradioactive nucleotide added). , NAD+; , NADH; •, ATP; x, NADP+; , NADPH, all photocross-linking with [32P]NAD+; , photocross-linking with [-32P]ATP in the presence of increasing concentrations of NAD+.

Flow Dialysis—Because photocross-linking experiments are not suitable for determining ligand binding constants accurately, we resorted for these determinations to the technique of flow dialysis (34). It allows accurate K_D determinations under the condition that this constant is less than or equal to that of the protein concentration, which was 30–50 µM for His¹⁰-VaKtrA. Fig. 3A shows an experiment in which the binding of [-³²P]ATP to His¹⁰-KtrA was measured. Compared with the situation without protein (open triangles), in the presence of His¹⁰-VaKtrA only about 20% of the added 30 µM of [-³²P]ATP appeared in the dialysate (closed triangles), indicating strong binding of the nucleotide to the protein. Subsequent additions of nonradioactive ATP gradually released the radioactivity from the protein (Fig. 3A). From these data a K_D value of 7 µM was calculated for the binding of ATP to His¹⁰-VaKtrA. Repetition of the experiment gave a K_D value of 9 µM ± 0.8 for three different His¹⁰-VaKtrA preparations (Fig. 3B and Table 1). A similar value was obtained with VaKtrA from which its His tag had been removed previously. In addition, the presence of 1 mM concentrations of any of the divalent cations Mg²⁺, Ca²⁺, Fe²⁺, Cu²⁺, Mn²⁺, Ni²⁺, or Co²⁺ did not influence ATP binding to His¹⁰-KtrA (results not shown).

Release of [-³²P]ATP by Other Nucleotides—In the experiments of Fig. 3, D–F and Table 1 we tested to what extent nonradioactive nucleotides other than ATP released [-³²P]ATP from His¹⁰-KtrA. First, we observed that concentrations of up to 700 µM nonradioactive NAD⁺ did not release any radioactivity (Fig. 3D), confirming that NAD⁺ does not bind with high affinity to His¹⁰-VaKtrA. By contrast, both AMP and NADH and a number of other nucleotides released some radioactivity from VaKtrA (Fig. 3, panels E and F, and results not shown). From these data we determined K_D (K_i) values for the different nucleotides according to the method described in the "Experimental Procedures" (Table 1). We conclude that the order of affinity of binding to His¹⁰-VaKtrA is ATP > ADP > FAD > AMP CTP > NADH > NAD⁺, GTP (Table 1).

View larger version (38K): [in this window] [in a new window]   FIGURE 3. Flow dialysis assay for the binding of [-32P]ATP to His10-VaKtrA. The upper chamber was filled with 1 ml of buffer containing 30–50 µM (about 0.75–1.25 mg/ml) of His10-KtrA and stirred slowly. At zero time 0.5–1 µCi/ml [-32P]ATP (10–30 µM ATP) (panels A, and D–F) or [32P]NAD+ (30 µM NAD+, panel C) was added to the upper chamber, and the amount of radioactivity in the dialysate flowing through the lower chamber was determined as a function of time. After the initial addition of radioactivity, increasing concentrations of nonradioactive nucleotides were added at the concentrations indicated in the individual panels, leading to release of the radioactive nucleotide from His10-KtrA and thereby to an increase of radioactivity in the dialysate. A, the addition of 30 µM [-32P]ATP followed by nonradioactive ATP. B, Scatchard plot for the binding of ATP to His10-KtrA calculated from the data of panel A. C, the addition of 30 µM [32P]NAD+ at zero time followed by increasing concentrations of nonradioactive NAD+. D, like A, but with the addition of increasing concentrations of NAD+ instead of ATP followed by two additions of ATP. E, like A, but with the addition of increasing concentrations of NADH instead of ATP followed by the addition of 50 µM ATP. F, like A, but with the addition of increasing concentrations of AMP instead of ATP. , controls without His10-VaKtrA; , assays with His10-VaKtrA.

VaKtrA Lacks ATPase Activity—Because VaKtrA binds ATP so avidly (Fig. 3), we tested whether the protein hydrolyzes ATP (36). Both in the absence and presence of divalent cations we failed to detect any ATPase activity (data not shown). The detection limit in this test was an activity of 0.4 nmol of ATP hydrolyzed·min^–1·mg^–1 of His¹⁰-VaKtrA.

ATP Promotes the Binding of KtrA to VaKtrB-His⁶—It was then investigated whether ATP affects the formation of the KtrAB complex. In a first experiment we used the overlay technique. Proteins from membranes containing overproduced VaKtrB-His⁶ (15) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. On this membrane we detected the protein with a monoclonal penta-His antibody (Fig. 5, lanes 2 and 4). Subsequently, a VaKtrB-His⁶-containing gel strip was incubated with VaKtrA from which its His¹⁰ tag had been removed. Binding of VaKtrA to VaKtrB-His⁶ was detected with a polyclonal anti-VaKtrA antiserum. In the absence of ATP some KtrA bound to VaKtrB-His⁶ (Fig. 5, lane 5). However, this binding was substantially enhanced by low concentrations of ATP (see Fig. 7, lanes 6–9). Comparison with the data from Fig. 3 showed that ATP stimulated binding of KtrA to KtrB with an affinity similar to that for binding of ATP to KtrA (Fig. 5A). Controls demonstrated that VaKtrA did not bind to bovine serum albumin in the presence of absence of ATP and that 100 µM nucleotides NAD⁺, NADH, ADP, AMP, CTP, or FAD did not promote the binding of KtrA to VaKtrB-His⁶ (data not shown).

Because VaKtrB-His⁶ is expected to be at least partially unfolded on the nitrocellulose membrane, we investigated whether ATP also promotes the binding of KtrA to natively folded VaKtrB-His⁶. For this purpose we bound the n-dodecyl -D-maltoside-solubilized VaKtrB-His⁶ to Ni²⁺-agarose. After washing away contaminating proteins with low imidazole concentrations, the Ni²⁺-agarose-VaKtrB-His⁶ complex was incubated with VaKtrA in the presence or absence of 100 µM ATP (Fig. 6). Subsequently, non-bound VaKtrA was washed away, and VaKtrB-His⁶ was eluted with 100–250 µM imidazole. The presence of VaKtrB-His⁶ and VaKtrA in the eluate was detected with the same antibodies as in the experiment of Fig. 5. Whereas in the absence of ATP little VaKtrA co-eluted with VaKtrB-His⁶ (Fig. 6, lanes 1 and 2), in its presence a considerable amount of VaKtrA-VaKtrB-His⁶ complex was obtained from the column (Fig. 6, lanes 3 and 4), demonstrating again that ATP promotes complex formation between the two VaKtr proteins.

VaKtrAB Requires ATP and a High Membrane Potential for in Vivo Activity in E. coli—It was then examined whether VaKtrAB requires ATP for activity in vivo. We have established that plasmid-encoded Ktr activity measurements can be done in E. coli (4, 12, 15, 37). By using an E. coli atpBEFHAGDC strain, effects on transport of a high membrane potential (, as the main component of the proton motive force) and ATP can be distinguished from each other (8, 38, 39). The experiments were done with strain LB692, which combines the atp trait with the feature that it possesses a very low K⁺ transport activity because its intrinsic K⁺-uptake systems Kdp, Trk^H, Trk^G, and Kup are inactive (8).

First we examined the effect of the protonophore 2,4-dinitrophenol on VaKtrAB activity. At increasing concentrations it decreased the K⁺ transport activity to a low value under conditions at which the ATP level in the cells remained high (Fig. 7), indicating that ATP alone is not the energy source for K⁺ transport via KtrA. The presence of 1 mM 2,4-dinitrophenol increased the ATP concentration in the cells of the atp strain (Fig. 7B), presumably because uncontrolled respiration stimulates glycolysis and thereby ATP synthesis in these cells (40). Further experiments were done at this 2,4-dinitrophenol concentration.

View larger version (61K): [in this window] [in a new window]   FIGURE 4. ATP induces a conformational change in VaKtrA. His10-VaKtrA was subjected to limited proteolysis by trypsin in the absence (A) or presence of 1 mM of NAD+ (B), NADH (C), or ATP (D). The peptide pattern was analyzed as a function of time. Identified protein bands: 1, VaKtrA with residue H21 of His10-VaKtrA as N-terminal extension; 2, the dimer of 2; 3 and 4, VaKtrA fragments, which were formed only in the presence of ATP. Lane 1, control, before the addition of trypsin; lanes 2–5, the reaction was terminated 0.5, 10, 30, and 60 min, respectively, after the addition trypsin inhibitor.

View larger version (39K): [in this window] [in a new window]   FIGURE 5. ATP promotes the binding of VaKtrA to VaKtrB-His6 on a nitrocellulose membrane. Membrane proteins from KtrB-His6-producing strain Rosetta2(DE3)/pEL903 were separated by SDS-PAGE in eight identical lanes and transferred to a nitrocellulose sheet. Subsequently, each of the 8 lanes was cut out from the sheet separately and incubated with 500 µg/ml purified VaKtrA in the presence of 0–100 µM ATP (lanes 4–9). VaKtrA and VaKtrB-His6 were detected with the anti KtrA serum and the monoclonal anti-penta-histidine serum, respectively. Lane 1, marker proteins; lane 2, control, no KtrA present, detection of VaKtrB-His6 with the anti-pentahistidine serum; lane 3, control, no VaKtrA present, incubation with the anti-KtrA serum; lane 4, incubation with KtrA in the absence of ATP, detection of VaKtrB-His6; lanes 5–9, incubation with KtrA in the presence of 0, 1, 5, 50, and 100 µM ATP, respectively, detection of VaKtrA.

View larger version (42K): [in this window] [in a new window]   FIGURE 6. ATP promotes the binding of VaKtrA to VaKtrB-His6 bound to a Ni2+-agarose column. Solubilized KtrB-His6 was bound to a Ni2+-agarose column as described under "Experimental Procedures." Subsequently the column was incubated with 500 µg/ml VaKtrA in the presence or absence of ATP. After some washing steps VaKtrB-His6 was eluted with 250 mM imidazole. VaKtrA and VaKtrB-His6 in the eluate were detected with the anti-KtrA serum and the monoclonal anti-pentahistidine serum, respectively. Lanes 1 and 2, VaKtrA retained by VaKtrB-His6 in the absence of ATP; lanes 3 and 4, the same in the presence of 100 µM ATP; lane 5, control, incubation of the column with VaKtrA in the absence of VaKtrB-His6; lanes 1 and 3, detection of VaKtrB-His6; lanes 2, 4, and 5, detection of VaKtrA.

View larger version (16K): [in this window] [in a new window]   FIGURE 7. K+ uptake via VaKtr requires . K+ uptake by K+-depleted cells of strain LB692/pKT84 (A) and their ATP contents (B) were determined as a function of time in the absence or presence of 2,4-dinitrophenol. The additions were 10 mM glucose at t =–30 min, 2,4-dinitrophenol at t =–5 min, 1 mM KCl at t = 0. , control; •, , and , 0.5, 1, and 2 mM 2,4-dinitrophenol, respectively. wt, wild type.

View larger version (22K): [in this window] [in a new window]   FIGURE 8. K+ uptake via VaKtr requires both ATP and . The effect of different energy sources on the uptake of K+ (A), [14C]glutamine (B), and [14C]proline (C )by K+-depleted cells of strain LB692/pKT84 and the ATP contents of these cells (panels D–F) were measured as a function of time. Substrate, 2,4-dinitrophenol, and K+ or radioactive amino acid were added at t =–30 min, t =–5, and t = 0 min, respectively. , substrate 10 mM glucose; •, substrate 10 mM glucose in the presence of 1 mM 2,4-dinitrophenol; , substrate 20 mM disodium succinate; , no substrate added.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Because at that time it was not known that a KtrA fragment binds ATP (17), we started our studies by characterizing the binding of NAD⁺ to the His-tagged KTN subunit His¹⁰-VaKtrA (Figs. 2 and 3). We also established that only parts of the C-terminal flexible domain of VaKtrA can be deleted with retention of function (Fig. 9). Therefore, we did our binding studies with the complete VaKtrA protein rather than with KTN protein fragments as in Roosild et al. (16) and Albright et al. (17). In accordance with (17), NAD⁺ bound with only low affinity to isolated His¹⁰-VaKtrA (Figs. 2 and 3), and more importantly, nonradioactive ATP prevented [³²P]NAD⁺ binding much better than did either nonradioactive NAD⁺ or NADH (Fig. 2). The flow-dialysis assay showed directly that [³²P]ATP binds with high affinity to His¹⁰-VaKtrA (K_D of 9 µM; Fig. 3, panels A and B). Because high affinity ligand binding to KtrA does not necessarily reflect the situation of ligand binding to the KtrAB complex, we tested whether ATP has a function in the formation of a KtrAB complex. Unlike other nucleotides, 10–100 µM ATP promoted the formation of this complex, and with these experiments we confirmed the existence of a KtrAB complex (Figs. 5 and 6; Ref. 17). Finally we established that K⁺ uptake via VaKtr requires ATP besides a high membrane potential when tested in an E. coli atp strain (Figs. 7 and 8).

The ligand-mediated conformational switch model is attractive for explaining the mechanism of K⁺ transport via the Ktr system (16). However, both the data of (17) and our results require its refinement in that ATP rather than NAD⁺/NADH is the regulating ligand. Our trypsin digestion results support the notion that upon binding ATP causes a conformational change in KtrA (Fig. 4). Under this condition a new peptide appears (peptide 4 in Fig. 4, panel D) of which its N terminus is located within putative helix G of KtrA (supplemental Fig. S1). This helix forms part of the flexible subunit interaction domain of KtrA, of which Roosild et al. (16) have proposed that upon ligand binding to the N-terminal ligand binding domain of KtrA, it changes its angle with the N-terminal domain. Our results that in the presence of ATP the tryptic fragment peptide 4 is formed indicate that ATP increases the accessibility of the subunit-interaction domain, allowing trypsin to cut behind residue Arg-¹³⁹ of VaKtrA.

View larger version (48K): [in this window] [in a new window]   FIGURE 9. Parts of the C-terminal flexible peripheral domain of KtrA are required for K+ transport activity of the VaKtr system. A, C-terminal deletions of ktrA in the ktrAB-containing plasmid pKT84 (4) were constructed, and their effects on K+ transport were examined as described under "Experimental Procedures"; see also the list of plasmids pMW130stop to pMW180stop in supplemental Table 1. BsKtrA refers to the 144-amino acid N-terminal KtrA fragment from B. subtilis for which crystal structures in complex with NADH and ATP are available (16, 17). B, detection of 35S-labeled VaKtrA (lane 1) and its C-terminal-truncated variants (lanes 2–9) in a minicell system; lane 2, VaKtrA1–179; lane 3, VaKtrA1–169; lane 4, VaKtrA1–164; lane 5, VaKtrA1–159; lane 6, VaKtrA1–154; lane 7, VaKtrA1–149; lane 8, VaKtrA1–139; lane 9, VaKtrA1–129.

Figs. 5 and 6 document that the actual role of ATP in the regulation of the Ktr system may lie in the promotion of the formation of the KtrAB complex. It could be that because of a lack of ATP binding in starving cells, KtrA dissociates from the membrane and thereby decreases the activity of VaKtrB to the very low value observed for this protein alone (15). This situation would parallel that of V-type ATPases, for which it known that the peripheral V₁ complex easily dissociates from the V_o complex in the membrane (44, 45) and that the presence of ATP prevents this dissociation.⁵ In addition, MgATP promotes the formation of a F₁ complex from the single subunits (46). Clearly, more work is needed to elucidate the mechanism by which ATP binding regulates the Ktr system.

Our results differ from those in Albright et al. (17) in that the affinity of ATP for binding to KtrA varies by a factor of more than 100. The cause of this difference may be severalfold, including species specificity (KtrA from B. subtilis (17) and V. alginolyticus (our work)), the use of a KtrA fragment (17) or the complete protein (our work), and the application of different techniques for measuring ATP binding (ATPS-BODIPY fluorescence (17) versus flow dialysis (our work).

Bacteria are known to contain cytoplasmic ATP concentrations of up to 5 mM (e.g. Ref. 47). Hence, it appears amazing that the K_D value for ATP binding to a KtrA fragment was with 600 nM (17) and 9 µM VaKtrA (Fig. 3 and Table 1) 5 to 3 orders of magnitudes lower than that of total ATP in the cell. However, it should be taken into consideration that bacteria also contain high Mg²⁺ concentrations and that, therefore, because of formation of the Mg²⁺-ATP complex the free ATP concentration in the cells is much lower than that of total ATP. In addition, a comparison with other systems shows that a K_D value of 600 nM to 9 µM for binding of Mg²⁺-ATP is not unusually low. For instance, the isolated regulatory subunit and the isolated catalytic subunit of the F₁ ATPase from E. coli bind Mg²⁺-ATP with K_D values of 0.1 and 40 µM, respectively (Ref. 46 and the references cited Ref. 48). In addition, the three catalytic sites of the E. coli F₁ ATPase have K_D values for Mg²⁺-ATP of 1 nM, 1 µM, and 30 µM, respectively (49). It is also possible that the affinity for ATP binding to KtrA in the KtrAB complex is lower than that to KtrA alone. Finally, our data seem to exclude that the Ktr system functions as a K⁺-translocating ATPase, since first, VaKtrA failed to show any ATPase activity (results not shown), and second, in intact cells the presence of ATP alone did not lead to Ktr activity (Figs. 7 and 8).

	FOOTNOTES

 
^* This work was supported by the Deutsche Forschungsgemeinschaft SFB431, Teilprojekt P6. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental material.

This work is dedicated to E. C. Slater on the occasion of his 90th birthday.

² Present address: Institute of Biochemistry, University of Kiel, D-24098 Kiel, Germany.

³ Present address: Klinik und Poliklinik für Unfall-, Hand und Wiederherstellungschirurgie, University Clinics, University of Münster, D-48149, Münster, Germany.

¹ To whom correspondence should be addressed: Dept. of Microbiology, University of Osnabrück, D-49076 Osnabrück, Germany. Tel.: 541-969-2274; Fax: 541-696-2870; E-mail: kroening{at}biologie.uni-osnabrueck.de.

⁴ The abbreviations used are: Va, V. alginolyticus; , membrane potential (internally negative); ATPS, adenosine 5'-O-(thiotriphosphate).

⁵ Dr. Helmut Wieczorek, University of Osnabrück, personal communication.

	ACKNOWLEDGMENTS

 
We thank Eva Limpinsel for expert technical assistance, Henryk Strahl for generating supplemental Fig. 1, and Wolfgang Epstein for pointing out that it may be ATP rather than NAD⁺ or NADH that binds strongly to VaKtrA and for help with the manuscript.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Dinnbier, U., Limpinsel, E., Schmid, R., and Bakker, E. P. (1988) Arch. Microbiol. 150, 348–357[CrossRef][Medline] [Order article via Infotrieve]
2. Stumpe, S., Schlösser, A., Schleyer, M., and Bakker, E. P. (1996) in Handbook of Biological Physics, Volume 2: Transport Processes in Eukaryotic and Prokaryotic Organelles (Konings, W. N., Kaback, H. R., and Lolkema, J. S., eds) pp. 474–499, Elsevier Science B.V., Amsterdam
3. Record, T. M., Courtenay, E. S., Cayley, D. S., and Guttman, H. J. (1998) Trends Biochem. Sci. 23, 143–1484[CrossRef][Medline] [Order article via Infotrieve]
4. Nakamura, T., Yuda, R., Unemoto, T., and Bakker, E. P. (1998) J. Bacteriol. 180, 3491–3494[Abstract/Free Full Text]
5. Holtmann, G., Bakker, E. P., Uozumi, N., and Bremer, E. (2003) J. Bacteriol. 185, 1289–1298[Abstract/Free Full Text]
6. Rhoads, D. B., and Epstein, W. (1978) J. Gen. Physiol. 72, 283–295[Abstract/Free Full Text]
7. Parra-Lopez, C., Baer, M. T., and Groisman, E. A. (1993) EMBO J. 12, 4053–4062[Medline] [Order article via Infotrieve]
8. Harms, C., Domoto, Y., Celik, C., Rahe, E., Stumpe, S., Schmid, R., Nakamura, T., and Bakker, E. P. (2001) Microbiology 147, 2991–3003[Abstract/Free Full Text]
9. Schlösser, A., Hamann, A., Bossemeyer, D., Schneider, E., and Bakker, E. P. (1993) Mol. Microbiol. 9, 533–543[Medline] [Order article via Infotrieve]
10. Murata, T., Takase, K., Yamato, I., Igarashi, K., and Kakinuma, Y. (1996) J. Biol. Chem. 271, 10042–10047[Abstract/Free Full Text]
11. Kawano, M., Igarashi, K., and Kakinuma, Y. (1999) FEMS Microbiol. Lett. 176, 449–453[CrossRef]
12. Tholema, N., Bakker, E. P., Suzuki, A., and Nakamura, T. (1999) FEBS Lett. 450, 217–220[CrossRef][Medline] [Order article via Infotrieve]
13. Durell, S. R., Hao, Y., Nakamura, T., Bakker, E. P., and Guy, H. R. (1999) Biophys. J. 77, 775–789[Medline] [Order article via Infotrieve]
14. Durell, S. R., Bakker, E. P., and Guy, H. R. (2000) Biophys. J. 78, 188–199[Medline] [Order article via Infotrieve]
15. Tholema, N., Vor der Brüggen, M., Mäser, P., Nakamura, T., Schroeder, J. I., Kobayashi, H., Uozumi, N., and Bakker, E. P. (2005) J. Biol. Chem. 280, 41146–41154[Abstract/Free Full Text]
16. Roosild, T. P., Miller, S., Booth, I. R., and Choe, S. (2002) Cell 109, 781–791[CrossRef][Medline] [Order article via Infotrieve]
17. Albright, R.A., Vasquez-Ibar, J.-L., Kim, C. U., Gruner, S. M., and Morais-Cabral, J. H. (2006) Cell 126, 1147–1159[CrossRef][Medline] [Order article via Infotrieve]
18. Jiang, Y., Pico, A., Cadene, M., Chait, B. T., and MacKinnon, R. (2001) Neuron 29, 593–601[CrossRef][Medline] [Order article via Infotrieve]
19. Jiang, Y., Lee, A., Chen, J., Cadene, M., Chait, B. T., and MacKinnon, R. (2002) Nature 417, 515–522[CrossRef][Medline] [Order article via Infotrieve]
20. Roosild, T. P., Le, K. T., and Choe, S. (2004) Trends Biochem. Sci. 29, 39–45[CrossRef][Medline] [Order article via Infotrieve]
21. Kuo, M. M. C., Haynes, W. J., Loukin, S. H., Kung, C., and Saimi, Y. (2005) FEMS Microbiol. Rev. 29, 961–985[CrossRef][Medline] [Order article via Infotrieve]
22. Parra-Lopez, C., Lin, R., Aspedon, A., and Groisman, E. A. (1994) EMBO J. 13, 3964–3972[Medline] [Order article via Infotrieve]
23. Wierenga, R. K., Terpstra, P., and Hol, W. G. J. (1986) J. Mol. Biol. 187, 101–107[CrossRef][Medline] [Order article via Infotrieve]
24. Branden, C., and Tooze, J. (1991) Introduction to Protein Structure, Garland Publishing, Inc., pp. 144–152, New York and London
25. Parfenova, L. V., Crane, B. M., and Rothberg, B. S. (2006) J. Biol. Chem. 281, 21131–21138[Abstract/Free Full Text]
26. Stewart, G. S. A. B., Lubinsky-Mink, S., Jackson, C. G., Cassel, A., and Kuhn, J. (1986) Plasmid 15, 172–181[CrossRef][Medline] [Order article via Infotrieve]
27. Guzman, L.-M., Belin, D., Carson, M. J., and Beckwith, J. (1994) J. Bacteriol. 177, 4121–4130
28. Epstein, W., and Kim, B. S. (1971) J. Bacteriol. 108, 639–644[Abstract/Free Full Text]
29. Studier, F. W. (1991) J. Mol. Biol. 219, 37–41[CrossRef][Medline] [Order article via Infotrieve]
30. Klionsky, D. J., Brusilow, W. S. A., and Simoni, R. D. (1984) J. Bacteriol. 160, 1055–1060[Abstract/Free Full Text]
31. Nakamura, T., Yamamuro, Stumpe, S., Unemoto, T., and Bakker, E. P. (1998) Microbiology 144, 2281–2289[Abstract/Free Full Text]
32. Kimmich, G. A., Randles, J., and Brand, J. S. (1975) Anal. Biochem. 69, 187–206[CrossRef][Medline] [Order article via Infotrieve]
33. Carrol, S. F., Lory, S., and Collier, R. J. (1980) J. Biol. Chem. 255, 12020–12024[Abstract/Free Full Text]
34. Colowick, S. P., and Womack, F. C. (1969) J. Biol. Chem. 244, 774–777[Abstract/Free Full Text]
35. Fersht, A. (1985) Enzyme Structure and Mechanism, 2nd Ed., W.H. Freeman and Co., New York
36. Arnold, A., Wolf, H. U., and Ackermann, B. P. (1976) Anal. Biochem. 71, 209–213[CrossRef][Medline] [Order article via Infotrieve]
37. Matsuda, N., Kobayashi, H., Katoh, H., Ogawa, T., Futatsugi, L., Nakamura, T., Bakker, E. P., and Uozumi, N. (2004) J. Biol. Chem. 279, 54952–54962[Abstract/Free Full Text]
38. Berger, E. A. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 1514–1518[Abstract/Free Full Text]
39. Rhoads, D. B., and Epstein, W. (1977) J. Biol. Chem. 252, 1394–1401[Abstract/Free Full Text]
40. Bakker, E. P., and Randall, L. L. (1984) EMBO J. 3, 895–900[Medline] [Order article via Infotrieve]
41. Jung, H. (2001) in Microbial Transport Systems, (Winkelmann, G., ed) pp. 47–75, Wiley-VCH, Weinheim
42. Stumpe, S., and Bakker, E. P. (1997) Arch. Microbiol. 167, 126–136[CrossRef][Medline] [Order article via Infotrieve]
43. Neidhardt, E. C., Ingraham, J. L., and Schaechter, M. (1990) Physiology of the Bacterial Cell: A Molecular Approach, p. 312 Sinauer Associates, Inc., Sunderland, Massachusetts
44. Sumner, J.-P., Dow, J. A. T., Earley, F. G. P., Klein, U., Jäger, D., and Wieczorek, L. (1995) J. Biol. Chem. 270, 5649–5653[Abstract/Free Full Text]
45. Kane, P. M. (1995) J. Biol. Chem. 270, 17025–17032[Abstract/Free Full Text]
46. Dunn, S. D., and Futai, M. (1980) J. Biol. Chem. 255, 113–118[Free Full Text]
47. Schleyer, M., Schmid, R., and Bakker, E. P. (1993) Arch. Microbiol. 160, 424–431[CrossRef][Medline] [Order article via Infotrieve]
48. Senior, A. E. (1990) Annu. Rev. Biophys. Bioeng. 19, 7–41
49. Weber, J., and Senior, A. E. (2003) FEBS Lett. 545, 61–70[CrossRef][Medline] [Order article via Infotrieve]

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