From the Department of Biochemistry and Molecular Biology, Oregon
Health Sciences University, Portland, Oregon 97201-3098
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INTRODUCTION |
The Na,K-ATPase (3.6.1.37) is an integral membrane protein that is
responsible for maintaining ionic homeostasis in animal cells by
mediating the active translocation of sodium and potassium ions against
their electrochemical gradients across the plasma membrane. The ion
gradients produced by the sodium pump are important for cell
excitability and contractility, as well as for regulation of other
intracellular ion and solute concentrations. The current model for the
sodium pump cycle is conserved for other ion pumps, e.g. the
plasma membrane and sarcoplasmic reticulum calcium pumps, the gastric
hydrogen/potassium pump, and the Neurospora proton pump. These ion
pumps make up the enzyme class known as the P-type ATPases.
Although much information has been gathered about the kinetic mechanism
of the sodium pump, there is still rather little detail known about the
structure of the protein.
The sodium pump exists as a functional heterodimeric protein consisting
of a catalytic
-subunit (~110 kDa) and a smaller, glycosylated
-subunit (~55 kDa). Both subunits have been cloned, and the
primary structure has been determined in several isoforms from a
variety of species (1). However, even after extensive study for several
decades, information about the specific amino acids involved in
formation of the binding sites for physiological ligands is
rudimentary. Although the detailed topology of the
-subunit of the
sodium pump is still the subject of investigation, an overall consensus
on structure is emerging; it shows 10 transmembrane segments (3). All
the residues implicated with ATP binding thus far have been localized
to the major cytoplasmic loop, which is composed of about 430 amino
acid residues between transmembrane segments M4 and M5 (2). Moreover,
this loop contains four of the most highly conserved P-type ATPase
sequences (3). Much of the information suggesting the involvement of
various amino acid residues with pump function comes from chemical
modification experiments. Such studies have identified a number of
residues in the M4M5 loop thought to be involved in ATP binding. For
example, Lys480, Lys501, Gly502,
Asp710, Asp714 and Lys719 are all
modified by a variety of chemical agents in the absence of ATP but not
in its presence, suggesting a role in ATP binding (4). In subsequent
studies, some of these residues were changed via mutagenesis with
little or no effect on enzyme activity (1). However, measurements of
overall enzyme activity only provide information on whether the mutated
residue is absolutely essential for function, but they do not
necessarily address whether the residue is involved directly in ATP
binding. For example, mutation of Asp369 (i.e.
the site of phosphorylation), not surprisingly, abolishes enzyme
activity (5), but interestingly, mutation of this residue increases the
ATP binding affinity (6). Consequently, to determine whether a residue
is important for ATP binding one must directly measure ATP binding and
characterize its affinity. The only approach that will identify ATP
contact residues is to crystallize the protein in the presence of
substrate. Unfortunately, crystallographic analysis of integral
membrane proteins still remains difficult because generally applicable
strategies for obtaining x-ray quality crystals of these molecules do
not yet exist.
As an approach to achieving this aim, we describe the bacterial
production and purification of a soluble polypeptide corresponding to
the large cytoplasmic loop of the Na,K-ATPase. This soluble protein is
able to bind ATP, as evidenced by the ability of ATP to prevent
modification by both fluorescein 5'-isothiocyanate (FITC)1 and
2-[4'maleimidylanilino]napthalene-6-sulfonic acid (MIANS), two
fluorescent probes previously demonstrated to label the M4M5 loop of
the full-length Na,K-ATPase in an ATP-protectable fashion. In addition,
we used 2',3'-O-[2,4,6,-trinitrophenyl]adenosine 5'-triphosphate (TNP-ATP) fluorescence to estimate, by competition with
ATP, the ATP binding affinity to this segment of the sodium pump. A
preliminary report of some of this work was presented at the Eighth
International Meeting on the sodium pump (7).
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EXPERIMENTAL PROCEDURES |
Reagents, Media, and Bacterial Strains--
NaCl, ATP, ADP, AMP,
CTP, GTP, UTP, ethanol, ethidium bromide, bovine serum albumin,
ultrapure urea, 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside, and Tris base were purchased from
Sigma.
-mercaptoethanol, SDS, ammonium persulfate, Coomassie
Brilliant Blue R-250, DNA miniprep kit, and low molecular weight
standards were from Bio-Rad. 4-(2-Aminoethyl)-benzenesulfonyl
fluoride hydrochloride was from ICN. Acrylamide and bisacrylamide were
from Boehringer Mannheim. Rainbow gel electrophoresis standards were
from Amersham Pharmacia Biotech. FITC, MIANS, and TNP-ATP were from
Molecular Probes. Polyvinylidene difluoride electroblotting membrane
was from Millipore. Tryptone, granulated agar, and yeast extract were from Difco. Agarose and restriction endonucleases were from Life Technologies, Inc. The pCR-Script cloning kit and Pfu DNA
polymerase were from Stratagene. The pET-28 expression vector and
Escherichia coli BL21(DE3) cells were from Novagen.
Ampicillin and kanamycin were obtained from the University Hospital
Pharmacy (Oregon Health Sciences University). E. coli DH5
cells were a generous gift from Dr. Linda Kenney (Molecular
Microbiology and Immunology, Oregon Health Sciences University). DNA
miniprep kits, DNA gel extraction kits, IPTG, and His6
antibody were from Qiagen. DNA sequencing was perfomed at the core
facility at Oregon Health Sciences University.
Construction of the Protein Expression Vector pAN--
The
portion of the rat
1-subunit encoding the M4M5 cytoplasmic loop
(Lys354-Lys774) was amplified via polymerase
chain reaction (PCR) in the presence of 10 µM
oligonucleotide primers (shown below), 1.2 mM of the four
deoxynucleoside triphosphates, and 5 units of Pfu DNA
polymerase in 50 µl of the manufacturer's buffer. The template was
pGEM-rat
1, a generous gift from Dr. Robert Mercer (Washington
University, St. Louis, MO). Twenty-five PCR cycles (30 s at 94 °C, 1 min at 53 °C, and 2 min at 72 °C) were performed in a PTC-100
thermal cycler (MJ Research Inc.). Agarose gel electrophoresis of the PCR revealed a single DNA fragment of 1.2 kilobases.
The forward primer was as follows.
The reverse primer was as follows.
After gel purification of the PCR product, a blunt end ligation
was performed with the SrfI-digested pCR-Script
plasmid according to the manufacturer's protocol (Stratagene) (Fig.
1). (pCR-Script confers ampicillin resistance and contains a portion of
the lacZ gene encoding the
-galactosidase gene product
providing blue-white color selection of recombinant plasmids.) Calcium
competent (8) E. coli DH5
cells were transformed with the
ligation mixture, and positive colonies were selected on
LBamp agar plates containing 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside and IPTG. White colonies were
selected and grown overnight in LBamp, and the DNA was
isolated via minipreparation techniques (Bio-Rad and Qiagen DNA
miniprep kits). Restriction endonuclease mapping revealed positive
clones, which were subsequently sequenced to verify that no random
mutations occurred during the PCR. The cloned M4M5 loop was released
from pCR-Script by digesting with NdeI and EcoRI; these unique restriction sites were engineered into the oligonucleotide primers (see above). The 6-histidine fusion protein vector, pET-28 (Novagen), was also digested with EcoRI and NdeI.
A ligation reaction was performed after gel purifying both DNA
fragments at a ratio of 10:1 (insert to vector) using T4 DNA ligase (1 unit/10 ng of DNA) at 25 °C for 1 h. The cloned product, called
pAN, was subsequently transformed into competent DH5
cells and
selected by the kanamycin resistance conferred by pET-28. DH5
cells
have a higher efficiency for transformation with ligation reactions
than do BL21 cells. Therefore, we used DH5
cells for initial
transformations and long term storage of vectors as glycerol stocks,
whereas we preferred BL21(DE3) cells for protein expression.
Overexpression of a 6-Histidine-tagged M4M5 Loop--
The
constructed pAN expression vector was used to transform calcium
competent BL21 (DE3) cells. The E. coli transformants were
selected on LBkan (30 µg/ml) agar plates. A single colony was picked to grow overnight in 5 ml of LBkan, and this
culture was subsequently used to inoculate 1 liter of LBkan
containing a final concentration of 2% ethanol. After the culture grew
to an A600 of 0.8-1.0, 1 mM IPTG
was added to induce the synthesis of protein from the lac promoter and
grown further at 25 °C to an A of ~2.0.
Cells were then collected and suspended in 30 ml of a lysis buffer
containing 50 mM Tris, 100 mM NaCl, pH 8.0, with a hand-held homogenizer. A 10-mg quantity of lysozyme was added in
the presence of 150 µM 4-(2-aminoethyl)-benzenesulfonyl
fluoride hydrochloride (serine protease inhibitor), and the mixture was
incubated on ice for 30 min, with occasional plunging of the
homogenizer. After addition of 40 mg of deoxycholic acid, the
suspension was heated to 37 °C with constant stirring. Once the
suspension became viscous and difficult to stir, 200 µl of
deoxyribonuclease (1 mg/ml) was added, and the mixture was incubated at
25 °C until the suspension was no longer viscous (~30 min). A
final concentration of 1% Triton X-100 was added, and the cell lysate
was incubated for an additional 30 min at 25 °C. The soluble
fraction was separated from the membranous fractions by centrifugation
at 12,000 × g for 45 min at 4 °C. Expression of the
His6 loop was verified by running a sample of the soluble
cell lysate on a 10% Laemmli gel and electroblotting onto
polyvinylidene difluoride membrane (10 mM CAPS, pH 11.0, 45 min). The polyvinylidene difluoride membrane was screened with a 1:1000
dilution of a 6-histidine antibody (Qiagen) revealing a protein (~46
kDa) from the lysate of IPTG-induced cells only.
Purification of His6 Loop via Ni2+-Column
Chromatography--
A 4-ml slurry of His-Bind resin (nitriloacetic
acid-agarose, Novagen) was placed into a 5-ml column; after the resin
settled, a bed volume of 2 ml remained. The column was washed with 10 bed volumes of sterile deionized H2O and then charged with
nickel by adding 5 bed volumes of 50 mM NiSO4.
Unbound Ni2+ was washed away with 5 bed volumes of binding
buffer (5 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH 7.9). One-third of the soluble cell lysate
(~10 ml) was applied to the Ni2+-column. The column was
subsequently washed with 5 bed volumes of binding buffer to remove
nonspecifically bound E. coli proteins. The desired
His6 loop protein was eluted from the
Ni2+-column with a linear imidazole gradient from 50 to 400 mM in a buffer containing 500 mM NaCl and 20 mM Tris (pH 7.9). Twenty-five 1.5-ml fractions were
collected (0.5-0.75 ml/min flow rate, gravity flow) over 45-60 min
and analyzed for protein content by the method of Bradford (9).
Protein-containing fractions were further analyzed via
SDS-polyacrylamide gel electrophoresis (i.e. 10% Laemmli
gel). The desired product was routinely recovered over 5-6 fractions
ranging from ~125 to 250 mM imidazole. The purity varied
slightly between preparations; only preparations that were
95% pure
(based upon densitometry of the Coomassie-stained peptide bands)
were used for experimental measurements.
FITC and MIANS Modification Experiments--
An aliquot (10-20
µg) of the purified dog kidney Na,K-ATPase or the bacterially
produced His6 loop was incubated with either 5 µM FITC or 50 µM MIANS for 10 min at
25 °C in 20 µl of a buffer containing 50 mM Tris (pH
7.4), 2 mM EDTA, and 5 mM of either ATP, ADP,
AMP, CTP, GTP, or UTP as indicated in the corresponding figure (Fig. 3,
A and B). For experiments testing whether FITC or
MIANS could label denatured protein, the protein was incubated in the
presence of 1% SDS prior to the chemical treatment. The chemical
modification reactions were stopped by the addition of 40 µl of
Laemmli sample buffer containing (at a 1:1:1 ratio) 10% SDS, 8 M urea, and 0.1 M Tris (pH 8.8) and a 5% final
concentration of
-mercaptoethanol. The entire 60-µl sample volume
was loaded and run on a 12% SDS-polyacrylamide gel according to the
method of Laemmli (10). FITC-labeled bands were visualized under UV illumination (366 nm), and then proteins were fixed and stained with
0.1% Coomassie Brilliant Blue R-250 in 10% acetic acid.
TNP-ATP Binding to Isolated His6 Loop and Competition
by Adenine Nucleotides--
TNP-ATP binding to the His6
loop was performed essentially as described by Moczydlowski and Fortes
(18) for TNP-ATP binding to Na,K-ATPase with minor changes. Briefly,
fluorescence changes were measured in quartz cuvettes on a model
PTI-QM1 (Photon Technology International, Monmouth Junction, NJ) steady
state fluorometer. The excitation wavelength was 410 nm (5 nm width),
and the emission wavelength was 545 nm (2 nm width). Aliquots of a 1 mM TNP-ATP stock solution were titrated into a 1-ml
solution of 50 mM MOPS (pH 7.5) containing 100 µg of
His6 loop maintained at 25 °C. The same TNP-ATP
additions were made into buffer only. The difference between the
protein-containing and protein-free solution revealed the fluorescence
increases due to interactions between TNP-ATP and the His6
loop.
Competition with nucleotides was measured by titrating aliquots from a
25 mM stock of ATP, ADP, or AMP into a 1-ml solution of 50 mM MOPS (pH 7.5) containing 1 µM TNP-ATP and
10 µM His6 loop. The high protein to TNP-ATP
ratio was to ensure that essentially all of the TNP-ATP was bound prior
to the nucleotide additions. If there is a significant portion of
TNP-ATP in solution, it is difficult to detect the TNP-ATP that is
displaced from the His6 loop by the competing nucleotides.
Titrations of the TNP-ATP/His6 loop solution with buffer
were made to determine the fluorescence changes caused by dilution.
Subtraction of the dilution effect from the nucleotide titration
experiments revealed the fluorescence changes associated with the
nucleotide-induced TNP-ATP displacement from the His6
loop.
Circular Dichroism Measurements--
CD spectra were taken on a
JASCO J-500 A spectrophotometer. Measurements were made using a 0.1-mm
path length cell (Helma) at a constant temperature of 20 °C. Data
were collected on an IBM/PC-XT using the IF-2 interface; software was
provided by Jasco. Spectra and buffer baselines were the average of 10 scans, each recorded at 0.1-nm intervals, using a scanning rate of 5 nm/min and a 2-s time constant. The protein concentration was
determined by amino acid analysis and was approximately 2.0 mg/ml.
Before spectral deconvolution for secondary structure analysis, the
buffer baseline was subtracted and the resulting spectrum was smoothed using the program provided by Jasco. The secondary structure of the
His6 loop was computed using the singular-value and
variable-selection methods of Compton et al. (11).
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RESULTS |
Our goal was to characterize the isolated ATP binding domain of
the Na,K-ATPase. As a means to this end, we developed a method to
produce large quantities of a soluble peptide corresponding to the
cytoplasmic loop between transmembrane segments M4 and M5
(i.e. from Lys354 to Lys774, rat
1 subunit).
Overexpression and Purification of the His6
-Loop
Fusion Protein--
The plasmid pAN was constructed by inserting the
cDNA encoding the rat
1 M4M5 cytoplasmic loop into pET-28
multiple cloning site at the ndeI (5') and the
EcoRI (3') restriction site locations downstream from the
histidine coding sequence (Fig. 1). pAN
was used to transform a BL21(DE3) E. coli strain. For
induction of gene expression, cells were grown in 1 liter of
LBkan medium containing a final ethanol concentration of
2%, and fusion protein production was induced with IPTG. (The presence
of ethanol significantly increased the amount of fusion protein in the
soluble fraction.) This process routinely yielded significant
production of the His6 loop fusion protein (molecular mass,
~46 kDa) with approximately 40% associated with the soluble fraction
(Fig. 2). The His6 loop was
purified via a Ni-NTA affinity column (~20-25 mg/liter; Fig. 2).

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Fig. 1.
Schematic representation of pAN construction
for the expression of the His6 loop fusion protein.
The pAN expression vector was constructed via a two step procedure. A
cDNA fragment encoding the M4M5 cytoplasmic loop of the rat
Na,K-ATPase 1 subunit was obtained by PCR and initially inserted
into the screening vector pCR-Script (Stratagene). The construct was
excised from pCR-Script and cloned into pET-28 (Novagen).
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Fig. 2.
SDS-polyacrylamide gel electrophoresis
analysis of His6 loop expression and purification. A
Laemmli gel showing His6 fusion protein production in
E. coli BL21(DE3) cells. A single colony was grown overnight
and used to inoculate 1L of LBkan. Protein synthesis was
induced with 1 mM IPTG. Stds, molecular mass
standards (kDs); Uninduced, 100 µl of uninduced whole cell
growth pelleted and resuspended in Laemmli sample buffer;
Induced, 100 µl of induced whole cell growth pelleted and
resuspended in Laemmli sample buffer; Pellet, aliquot of
pelleted fraction after cell lysis; Supernatant, aliquot of
soluble fraction after cell lysis; Flow Through, aliquot of
soluble fraction after being applied to Ni2+-NTA column;
Eluate, His6 fusion protein eluted from the
Ni2+-NTA column with imidazole.
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FITC Labeling of the His6 Loop--
FITC is a
fluorescent amine-reactive molecule that labels Lys501 in
the purified Na,K-ATPase; this reaction is prevented by the simultaneous presence of ATP (12, 13). In this study, we used ATP
protection against FITC labeling as a tool to demonstrate that ATP
binds to His6 loop (Fig.
3A). It is clear that after incubation with 5 µM FITC, both the purified Na,K-ATPase
and the His6 loop were labeled by FITC. Moreover, when the
FITC incubation was performed in the presence of either 5 mM ATP or 5 mM AMP, only ATP (not AMP)
prevented FITC modification of both the intact purified sodium pump and
the His6 loop (Fig. 3A).

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Fig. 3.
FITC labeling of purified dog Na,K-ATPase and
His6 loop fusion protein. Both pictures are of the
same Laemmli gel. The picture on the left was taken under UV
illumination to show FITC fluorescence, and the picture on the
right was taken after Coomassie Brilliant Blue R-250
staining. A 10-µg quantity of purified His6 loop was
incubated with 5 µM FITC for 10 min at 25 °C in the
absence (Ctl) or presence of 5 mM of the
indicated nucleotide triphosphate. A, comparison of FITC
labeling of native Na,K-ATPase and His6 loop.
STDs, molecular mass standards; E, Ctl and
L, Ctl, FITC only; E, ATP and L, ATP,
FITC with 5 mM ATP; E, AMP and L,
AMP, FITC and 5 mM AMP. Clearly, ATP prevents FITC
labeling, whereas AMP is without effect. B, FITC labeling of
the His6 loop in the presence of various nucleotides. Both
ATP and ADP prevented FITC modification (E, ATP and E,
AMP), whereas AMP, CTP, GTP, and UTP were without effect (L,
Ctl, L, ATP, and L, AMP).
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To further investigate the nucleotide specificity of the
His6 loop, we tested the ability of several nucleotides to
protect against FITC modification (Fig. 3B). Purified
His6 loop was incubated with 5 µM FITC in the
presence of 5 mM ATP, ADP, AMP, GTP, and UTP. It is clear
that both ATP and ADP protected against FITC, whereas AMP, CTP, GTP,
and UTP were unable to prevent FITC modification. These data are in
agreement with the nucleotide specificity demonstrated for the purified
Na,K-ATPase, which has been shown to bind ATP and ADP with high
affinity, (14). Therefore, it appears that the His6 loop
retains the structural parameters that confer nucleotide specificity in
the intact enzyme.
Structural Analysis of the His6 Loop--
The
structural integrity of the His6 loop was directly
reflected by the structural requirement for FITC modification,
i.e. after we denatured the His6 loop with 1%
SDS, incubation with FITC failed to modify the peptide (Fig.
4). Similar results were observed when we
denatured purified dog Na,K-ATPase with 1% SDS (Fig. 4). These results
are not due to an anomalous effect of SDS on the FITC reaction; FITC
was also unable to modify the His6 loop after denaturation
with 6 M guanidine-HCl or by repeated freezing and thawing
(data not shown).

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Fig. 4.
FITC labeling of intact and denatured
His6 loop and purified dog Na,K-ATPase. Both pictures
are of the same Laemmli gel. The picture on the left was
taken under UV illumination to show FITC fluorescence, and the picture
on the right was taken after Coomassie Brilliant Blue R-250
staining. A 10-µg quantity of purified dog Na,K-ATPase or
His6 loop was incubated with 5 µM FITC for 10 min at 25 °C in the absence (Ctl) or presence
(ATP) of 5 mM ATP. In addition, some samples
(L, SDS, L, SDS, ATP, and E, SDS) were denatured
with 1% SDS prior to FITC modification. STDs, molecular
mass standards. It is evident that denaturation prevents FITC labeling
of both the His6 loop and native Na,K-ATPase.
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More directly, the overall secondary structure of the His6
loop fusion protein was estimated by CD spectroscopy. As shown in Fig.
5, the resultant spectrum, which
summarizes the mean residue molar ellipticity as a function of
wavelength, exhibits a maximum at 188 nm and two minima, one at 206 nm
and the other at 221 nm. The spectrum is the average of 10 scans, the
general shape of which did not change. Analysis of the CD spectrum of
the His6 loop, calculated using the singular-value
decomposition method (11), predicts secondary structural elements
distributed approximately as 23%
-helix, 23% antiparallel
-sheet, 4% parallel
-sheet, 19%
-turn, and 32% random coil.
The total of the fractions in this method is not constrained to be
100%, but rather should lie between 95 and 105%. The observation that
the components sum to close to 100% indicates that the fusion protein
is highly structured as assessed by this technique.

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Fig. 5.
Circular dichroism spectrum of the
His6 loop fusion protein. CD spectrum of
His6 loop in 50 mM sodium phosphate, pH = 7.9.
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MIANS Labeling of the His6 Loop--
Recently, the
fluorescent sulfhydryl reagent MIANS was shown to inactivate the dog
kidney Na,K-ATPase by specifically labeling a cysteine residue in the
M4M5 loop of the Na,K-ATPase (15). Both MIANS labeling and enzyme
inactivation were prevented by the simultaneous presence of
ATP.2 In this study, we
tested whether ATP was able to protect against MIANS modification of
the His6 loop. Indeed, ATP, but not AMP prevented MIANS
labeling of the bacterially produced His6 loop (Fig.
6). However, unlike FITC, MIANS does
label the denatured peptide, but ATP no longer protects against its
modification (Fig. 6). The simplest explanation for a lack of ATP
protection is that there is no longer significant structure to bind
ATP.

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Fig. 6.
MIANS labeling of intact and denatured
His6 loop. Both pictures are of the same Laemmli gel.
The picture on the left was taken under UV illumination to
show MIANS fluorescence, and the picture on the right was
taken after Coomassie Brilliant Blue R-250 staining. A 10-µg quantity
of purified His6 loop was incubated with 50 µM MIANS for 10 min at 25 °C in the absence
(Ctl) or presence (ATP and AMP) of 5 mM ATP or AMP. In addition, some samples (SDS)
were denatured with 1% SDS prior to the MIANS reaction.
STDs, molecular mass standards. MIANS readily modified the
His6 loop in the absence of nucleotides or in the presence
of AMP, whereas ATP completely prevented modification. Unlike FITC
(Fig. 5), MIANS covalently modified the denatured protein, and ATP was
without effect under denaturing conditions.
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TNP-ATP Binding to the His6 Loop and Competition with
Nucleotides--
The interactions of the His6 loop and
TNP-ATP were studied. This fluorescent ATP analog has been shown to
bind with high affinity to the native Na,K-ATPase, as well as to other
members of the P-type ATPase family (17-19). TNP-ATP is a useful
fluorescent probe for studying the nucleotide binding site because its
fluorescence changes significantly on binding. When an aliquot of
His6 loop was added to a solution containing 5 µM TNP-ATP, it resulted in an enhancement of the TNP-ATP
fluorescence consistent with TNP-ATP binding to the engineered protein
(Fig. 7). The addition of ATP to a
solution containing His6 loop and TNP-ATP resulted in a
decrease in the fluorescent signal, as would be expected if ATP
displaced TNP-ATP from the His6 loop (Fig. 7).

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Fig. 7.
Recordings of the raw data for TNP-ATP
fluorescence. Excitation and emission wavelengths were 410 and 545 nm, respectively. Lane 1, 5 µM TNP-ATP in 50 mM MOPS buffer (pH 7.5) alone. Lane 2, addition
of purified His6 loop (final concentration, 10 µM). 20 data points were collected per second, and the
average of all the points from each group were used in subsequent
experiments. Lanes 3-5, increasing ATP concentrations
(lane 3, 300 µM ATP; lane 4, 600 µM ATP; lane 5, 2000 µM ATP).
There was a significant increase in the fluorescence intensity of
TNP-ATP after the addition of protein, consistent with TNP-ATP binding
to the His6 loop. Also, subsequent additions of ATP compete
with bound TNP-ATP, causing a decrease in the fluorescence signal.
These data are from a single experiment in which ATP decreased the
TNP-ATP fluorescence by ~80%. Over the course of all our
experiments, the maximal TNP-ATP fluorescence change ranged from 30 to
80% and was typically between 40 and 50%.
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The apparent binding affinity of the His6 loop for TNP-ATP
was determined by titrating small aliquots of TNP-ATP into a solution containing His6 loop. Plotting the fluorescence change
against [TNP-ATP] showed that the binding of TNP-ATP to the
His-tagged construct was composed of two parts: a saturable component
and a nonsaturable component (Fig.
8A). Subtraction of the
nonsaturable component from all the data revealed the fluorescence
changes due to specific TNP-ATP binding (Fig. 8B). The
apparent affinity of the His6 loop for TNP-ATP was ~3
µM (Table I).

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Fig. 8.
Nucleotide binding to the His6
loop fusion protein. The enhanced extrinsic fluorescence of
TNP-ATP upon binding to the His6 loop was observed at 545 nm after excitation at 410 nm. TNP-ATP was added in increments to a 50 mM MOPS solution containing His6 loop (100 µg/ml, pH 7.5) and the fluorescence intensity measured after each
addition. The data are expressed as the percentage of change in
fluorescence intensity. The fluorescence signal in the absence of
TNP-ATP (i.e. the His6 loop in MOPS buffer) and
the contribution from free TNP-ATP were subtracted from all the data
points. A, total change in fluorescence intensity as a
function of [TNP-ATP]. Data were fit to the equation: % change = (a · [TNP-ATP]/b + [TNP-ATP]) + (c · [TNP-ATP] + d), where a = maximal
fluorescence change, b = Kd app for TNP-ATP,
c = slope of the nonsaturable binding component, and
d = y intercept of the nonsaturable binding
component. B, change in fluorescence due to specific binding
of TNP-ATP. The nonsaturable linear component in A was
subtracted from all the data points and replotted in B as a
function of [TNP-ATP]. Data were fit to the equation: a
· [TNP-ATP]/b + [TNP-ATP]. The mean ± S.E. for
the Kd from three separate experiments are shown in
Table I.
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Table I
Apparent affinities of nucleotides for the purified His6 loop
Excitation and emission wavelengths were 410 and 545 nm, respectively.
Experiments were performed as described under "Experimental
Procedures." The Kd values were taken from at
least three separate experiments identical to those shown in Figs. 8
and 9. In addition, the measurements were performed on at least three
different His6 loop preparations for each nucleotide.
Fluorescence measurements made in the absence of protein showed that
nucleotides did not alter the free TNP-ATP fluorescence.
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The apparent affinities for ATP, ADP, and AMP were determined from
their ability to displace bound TNP-ATP from the His6 loop. For these experiments, a 10-fold molar excess of the His6
loop protein compared with TNP-ATP was used to ensure that all
(i.e. >99%) of the TNP-ATP was bound and that the
contribution from the nonsaturable TNP-ATP binding component was zero
(20). The competition of TNP-ATP binding by nucleotides is evidenced by a decrease in fluorescence intensity upon the release of TNP-ATP from
the His6 loop. A plot of [nucleotide] versus
percentage of change in fluorescence from a typical experiment is shown
in Fig. 9. The apparent
Kd values for ATP and ADP were ~350
µM and ~550 µM, respectively (Table I).
AMP showed no saturable binding in the concentration range examined.
For each nucleotide, the binding experiments were performed on three
separate preparations of His6 loop with comparable
results.

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Fig. 9.
Competition of TNP-ATP binding by adenine
nucleotides. The fluorescence of His6 loop bound
TNP-ATP was observed at 545 nm after excitation at 410 nm. Aliquots
from concentrated solutions of either ATP (A), ADP
(B), and AMP (C) were then added to mixture, and
the fluorescence changes were monitored. The decrease in fluorescence
due to dilution were subtracted from the data. The change in
fluorescence due to nucleotide binding was plotted against the
concentration of the corresponding nucleotide. Data were fit to the
same equation as in Fig. 8B. The mean ± S.E. for the
Kd from three separate experiments are shown in
Table I. Measurements were also made in the absence of protein, and no
effects of the nucleotides were observed on the free TNP-ATP
fluorescence.
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DISCUSSION |
We have produced a soluble polypeptide by bacterial overexpression
that is identical in sequence with the central loop of the rat
1-subunit. We have shown that the peptide binds ATP and ADP, but not
other nucleoside triphosphates, with the same specificity as native
Na,K-ATPase. This highly ordered peptide also shows labeling and
protection reactions characteristic of the intact Na,K-ATPase.
Chemical modification experiments (4, 2) of the Na,K-ATPase, as well as
site-directed mutagenesis studies (1), suggest that several amino acids
located between the fourth and fifth putative transmembrane segments
participate in the coordination of ATP. However, in some instances, the
data generated from these two methods appear to be contradictory. For
example, labeling Lys501 with a number of reagents
(e.g. FITC, SITS, and
N-(2-nitro-4-isothiocyanophenyl)-imidazole) completely
inactivates the enzyme; the prior binding of ATP prevents both
modification and inactivation, consistent with Lys501
playing a role in ATP binding. However, when Lys501 was
changed to methionine via site-directed mutagenesis (21), the enzyme
retained activity, demonstrating that Lys501 is not
essential for enzyme activity. How could Lys501 participate
in ATP binding and yet not be required for enzyme activity? It is
possible that Lys501 is one of several residues that form
the ATP binding pocket. Loss of a single contact residue might result
in a lower ATP affinity, but not in a loss of ATP binding. Therefore,
in the presence of saturating ATP, Na,K-ATPase activity might remain
normal. However, attaching a chemical reagent to a contact residue or a
nearby residue not only removes that residue from coordination but also occupies space in the vicinity of that residue; thus, modification of
Lys501 may prevent ATP binding via steric factors.
Consequently, it is becoming readily apparent that a detailed
three-dimensional structure is needed to conclusively identify the
contact sites for ATP and to adequately describe its binding
pocket.
To approach such experiments, the major cytoplasmic domain between M4
and M5 of the Na,K-ATPase was overexpressed in E. coli. Similar approaches have been employed by other laboratories to isolate
the ATP binding domains of the yeast proton pump (22), the sarcoplasmic
reticulum calcium pump (23), the sodium pump (24), and the cystic
fibrosis transmembrane conductance regulator (25). The cDNA from
the rat
1 subunit, encoding 420 amino acids from
Lys354-Lys774, was cloned into a fusion
protein vector (His6 tag, pET-28) and transformed into
E. coli. This method routinely yields approximately 20 mg of
pure soluble peptide per liter of cell culture. We demonstrated that
this expressed and purified peptide 1) retains an ordered structure, 2)
can be labeled by both FITC and MIANS, 3) binds both ATP and ADP, and
4) binds the fluorescent ATP analog, TNP-ATP. Experiments are currently
under way to crystalize the His6 loop.
Structural Analysis of the His6 Loop--
Detailed
structural topology of the sodium pump awaits three-dimensional x-ray
crystallographic analysis. However, some assessments have been made
that suggest an
-subunit structure with 10 transmembrane segments.
Also, all the residues associated with ATP binding have been localized
to the major cytoplasmic loop between transmembrane segments M4 and M5
(2). Furthermore, the residues thus far implicated in nucleotide
binding among all the members of the P-type ATPase family have been
assigned to this cytosolic domain. Structural models of the nucleotide
binding pocket of P-type ATPases have been proposed based upon sequence
homology with different kinases (e.g. adenylate kinase and
phosphoglycerate kinase; see Ref. 26). According to these models, the
large cytoplasmic loop is divided into three domains: a phosphorylation
domain, a nucleotide binding domain, and a central domain. We
calculated the
-helix and
-sheet content of the nucleotide
binding domain portion (Arg524-Ile654) of the
sheep Na,K-ATPase cytoplasmic loop from the data previously reported
(26). According to their predictions using the method of Chou and
Fasman (27), we obtained a composition of about 35%
-helix and
22.5%
-sheet. These values agree reasonably well with our current
findings of 23%
-helix and 27%
-sheet for the secondary
structure of the bacterially expressed M4M5 loop. The difference in
-helical content may suggest that the phosphorylation domain and the
central domain (included in our analysis and not in that of Taylor and
Green (26)) have significantly less helical structure than does the
nucleotide binding domain. Alternatively, it may simply reflect the
difference between CD analysis (this study) and primary structure
comparison (26) or a less than completely retained structure of our
His6 loop compared with native intact Na,K-ATPase.
Nucleotide Protection against Chemical Modification of the
His6 Loop--
For almost two decades, it has been known
that FITC irreversibly inhibits the Na,K-ATPase in an ATP-protectable
manner (28). The site of FITC modification was identified as
Lys501 (12, 13). Considerable evidence suggests that
Lys501 resides in the ATP binding site of the sodium pump;
this evidence includes the following: 1) ATP prevents modification of
Lys501 (28), 2) the reactivity of Lys501 is
sensitive to cation binding in a way similar to the effect that cation
occupancy has on direct ATP binding (29-31; 15), and 3) an equivalent
lysine residue exists in a conserved sequence among most members of the
P-type II ATPase family (3).
In a fashion similar to the intact Na,K-ATPase, ATP and ADP (but not
AMP) protect against FITC modification of our purified His6
loop (Fig. 3). Moreover, nucleotides that do not bind with high
affinity to the native enzyme also do not protect the His6 loop from FITC modification (Fig. 3). These findings demonstrate that
the His6 loop has folded sufficiently to allow formation of
a nucleotide binding pocket that shows selectivity. Indeed, denaturing
the His6 loop prior to FITC treatment results in no FITC
labeling; the same finding was observed with the native Na,K-ATPase (Fig. 4). Thus, it appears that the selective reactivity of
Lys501 toward FITC is a product of the special environment
in the folded central loop. The appropriate folding of this ATP binding
loop generates a highly reactive lysine at position 501. This is in contrast with the behavior of MIANS and Cys577 (see
below).
We have observed that ATP, but not AMP, can also protect the loop
against modification by the sulfhydryl reagent MIANS (Fig. 6).
Recently, MIANS has been shown to modify a specific cysteine residue in
the large cytoplasmic loop of purified Na,K-ATPase; ATP protects
against MIANS modification. Proteolytic digestion and N-terminal amino
acid sequencing has identified the MIANS-modified residue as either
Cys549 or Cys577.2 ATP protection
against modification at Lys501 and Cys577 (or
Cys549) suggests that a compact folding of the loop may
bring the distant segments of the peptide together. It is interesting,
however, that after denaturation of this loop, modification still
occurs with MIANS, but such modification is no longer affected by the simultaneous presence of ATP.
Nucleotide Binding Affinity--
The sodium pump and most other
P-type ATPases have a complex ATP dependence; a high affinity ATP
effect and a low affinity ATP effect. In the sodium pump,
phosphorylation of E1 to E1~P requires Na, Mg
and ATP. The Km value for Na-ATPase
(Km for ATP
1 µM) agrees well with
the measured ATP binding affinity (32, 33). At low ATP concentrations,
the rate of hydrolysis is slow and limited by the release of potassium
(34). However, higher ATP concentrations (Km(ATP)
100 µM) facilitate the deocclusion of potassium. This low
affinity effect is seen as the Km for ATP under (Na + K)-ATPase conditions. ATP activation of the sodium pump, with both
high and low apparent affinities, has been interpreted as being due to
either two distinct ATP sites or to a single ATP binding region that
alters its affinity in different pump conformations.
Using the fluorescent ATP analog, TNP-ATP, we were able to estimate the
ATP affinity of the His6 loop. In experiments designed to
determine the His6 loop affinity for TNP-ATP, we discovered that there were two components to the binding of this ATP analog, a
saturable and nonsaturable component. Hellen and Pratap (20) reported a
similar two-component binding curve for TNP-ATP binding to the native
Na,K-ATPase. Subtracting the nonspecific binding from the data,
revealed a specific saturable component with an apparent
Kd of ~3 µM for TNP-ATP (Fig.
8B; Table I). This value is about an order of magnitude
higher than the Na,K-ATPase binding affinity for TNP-ATP (~0.5
µM at 25 °C; Ref. 17), i.e. it appears to
represent a low affinity site.
An affinity of 300-400 µM was found for ATP by
competition and displacement of TNP-ATP (Table I). This value is in
reasonable agreement with the low ATP affinity for the
E2(K2) state of the native Na,K-ATPase
associated with potassium deocclusion. In addition, the expressed
domain has a similar affinity (Kd 500-600 µM; Table I) for ADP. This is consistent with the
observation that in the low affinity E2-state of the sodium
pump, ADP has been shown to facilitate potassium deocclusion and
transport (16, 35-36). AMP concentrations up to 3 mM were
not sufficient to displace TNP-ATP (Fig. 9C), and
concentrations of up to 5 mM were unable to protect against
FITC modification (Fig. 3).
Recently, the nucleotide binding domains of the sarcoplasmic reticulum
calcium pump (23), the yeast proton pump (22), and the sodium pump (24)
have been expressed in E. coli. In the sodium pump-expressed
domain, ATP binding was demonstrated by measuring MgATP protection
against photolabeling with 2-N3-ATP32; however,
Mg alone appeared to protect as well as MgATP (24). All our experiments
were performed in the absence of magnesium; thus, the effects are
solely due to the nucleotides themselves. ATP binding to expressed
loops from the proton pump and sarcoplasmic reticulum calcium pump was
estimated by ATP competition of TNP-ATP, as in this study. The
affinities for TNP-ATP binding to the calcium pump and proton pump
domains were 2 and 6 µM, respectively, similar to our
value of ~3 µM for the sodium pump domain. In contrast, the ATP affinity reported for the proton pump domain was ~3
mM (22), significantly different from that reported for the
calcium pump domain (~200 µM; Ref. 23) and here for the
sodium pump domain (~300 µM; Table I). The considerably
lower affinity for the proton pump ATP binding domain may be due to an
inherent property of the domain or possibly because the proton pump
construct was a glutathione S-transferase fusion protein,
whereas the calcium pump and sodium pump constructs were both
His-tagged proteins. Indeed, we were unable to successfully measure the
ATP affinity for the glutathione S-transferase version of
our construct even though we were able to demonstrate that ATP
protected against FITC modification. It turned out that ATP (5 mM) also protected against FITC labeling of glutathione
S-transferase
alone.3
It appears, then, that expression of the isolated central loops of
these P-type ATPases produces a protein that is able to selectively
bind ATP (or ADP) but with an affinity close to that seen in
E2 forms. Because the isolated loop has considerable
secondary structure, it seems reasonable to suppose that the high
affinity binding form is generated by interactions with other parts of the ATPase (probably the cation binding domains). One way of modeling these changes in the ATP binding site would be to suppose that there
are two states or forms, an R (relaxed) form and a T (tense) form, the
R form having lower affinity for ATP and the T form, higher affinity
(<1 µM). The changes in structure of the intact protein
that we identify as E1 (high sodium affinity and high ATP
affinity) and E2 (high potassium affinity and low ATP
affinity) are mirrored by changes in T and R forms, respectively, in
the ATP binding loop. It is interactions between the ATP binding domain in the loop and other segments of the protein that hold the loop in the
tense form, which has a high affinity for ATP. In isolation, the loop
is not constrained by these interactions, and a relaxed (R) form exists
with low substrate affinity.
The present study of ATP binding to the purified M4M5 loop of the
Na,K-ATPase provides a basis for future mutagenesis studies of the
residues thought to play a role in ATP binding. Furthermore, the
ability to obtain large quantities of pure soluble protein makes this
method valuable for detailed structural analyses of the wild-type and
mutant ATP binding domains of P-type ATPases.
We are grateful to Sylvia Daoud, Jeremy
Holden, and Jeremy Johnston for excellent technical assistance. In
addition, we thank Dr. Linda Kenney for insight and advice during the
cloning of both the glutathione S-transferase and
His6-tagged fusion proteins. We thank Dr. David Farrens for
helpful suggestions with the fluorometry experiments and Dr. Susan
Thornewell for helpful comments throughout this work. Dr. Hans Peter
Bachinger is gratefully acknowledged for help, both practical and
intellectual, with the CD measurements and interpretation.