Tài liệu Báo cáo khoa học: The phosphatase activity of the isolated H4-H5 loop of Na+/K+ ATPase resides outside its ATP binding site docx

The phosphatase activity of the isolated H
4
-H
5
loop of Na
+
/K
+
ATPase
resides outside its ATP binding site
Rita Krumscheid
1
,Ru¨ diger Ettrich
2
, Zofie Sovova
´
2
, Kla
´
ra Sus
ˇ
a
´
nkova
´
3
, Zdene
ˇ
kLa
´
nsky

´
3
,
Kater
ˇ
ina Hofbauerova
´
3,4
, Holger Linnertz
1
, Jan Teisinger
3
, Evz
˘
en Amler
3
and Wilhelm Schoner
1
1
Institute of Biochemistry and Endocrinology, Justus-Liebig-University Giessen, Germany;
2
Laboratory of High Performance
Computing, Institute of Physical Biology USB and Institute of Landscape Ecology ASCR, Nove
´
Hrady, Czech Republic;
Institutes of
3
Physiology and
4
Microbiology, Czech Academy of Sciences, Prague, Czech Republic

The structural stability of the large cytoplasmic domain (H
4
-
H
5
loop) of mouse a
1
subunit of N a
+
/K
+
ATPase (L354–
I777), the number a nd the location of its binding sites for
2¢-3¢-O-(trinitrophenyl) adenosine 5¢-triphosphate (TNP-
ATP) and p-nitrophenylphosphate (pNPP) were investi-
gated. C- and N-terminal shortening revealed that neither
part of the phosphorylation (P)-domain are necessary for
TNP-ATP binding. There is no indication of a second ATP
site on the P -domain of the isolated loo p, e ven though
others reported previously of its existence by TNP-N
3
ADP
affinity labeling of t he full enzyme. F luorescein isothio-
cyanate (FITC)-anisotropy measurements reveal a consid-
erable stability of the nucleotide (N)-domain suggesting that
it may not undergo a substantial conformational change
upon ATP binding. The FITC modified loop showed only
slightly diminished phosphatase activity, most likely due to a
pNPP site on the N-domain around N398 whose mutation
to D reduced the phosphatase activity by 50%. The amino

acids forming this pNPP site (M384, L414, W411, S400,
S408) are conserved in the a
1)4
isoforms of Na
+
/K
+
ATPase, whereas N398 is only conserved in the vertebrates’
a
1
subunit. The phosphatase activity of the isolated H
4
-H
5
loop was neither inhibited by ATP, nor affected by mutation
of D369, which is phosphorylated in native Na
+
/K
+
ATPase.
Keywords:ATPase;H
4
-H
5
loop; p-nitrophenylphosphate;
protein expression; TNP-ATP.
The Na
+
/K
+

ATPase (EC 3.6.3.9) or sodium pump carries
out the coupled extrusion and uptake of Na
+
and K
+
ions
across plasma membranes of mammalian cells. The enzyme
is a heterodimer of a 100 kDa catalytic subunit and a
heavily glycosylated b subunit of about 55 kDa [1–3].
Ouabain, recently recognized as a m ammalian s teroid
hormone [4], uses the sodium pump in the nanomolar
concentration range as a signal transducer [5] but inhibits it
at higher (toxic) concentrations [1–3]. The ion pumping
process is connected to a reaction cycle model with
conformational changes of the catalytic a subunit. Such
changes become visible amongst others in the Na
+
dependent generation of an aspartyl (D369) phosphointer-
mediate with different sensitivities towards the reaction
product ADP or the second transport substrate K
+
.The
observation of high and low affinity ATP sites with
approximate K
d
values of 1 l
M
(E
1
ATP site) and 200 l

M
(E
2
ATP site) in the membrane-embedded Na
+
/K
+
ATPase, and the finding that reaction inert MgATP
complex analogues such as Cr(H
2
O)
4
ATP and
Co(NH
3
)
4
ATP may react specifically with these ATP sites
[6] as well as the complex k inetic s with the fluore scent
2¢(3¢)-O-(6-N¢,N¢-dimethylaminonaphthalenesulfonyl)ATP
(DANSyl-ATP) [7,8], lead to the suggestion that high and
low affinity ATP sites coexist and that they interact during
catalysis. Consistent with this conclusion is the finding that
the activity of a K
+
activated phosphatase, which represents
a partial function of the ATP site [9–12], was almost
unaffected by the blockade of the ATP site due to
modification of K501 with fluorescein isothiocyanate
(FITC) [13], but was l ost when t he enzyme additionally

reacted with Co(NH
3
)
4
ATP [14,15] or erythrosin isothiocy-
anate [16]. The latter binds to C549 within the nucle otide
(N)-domain of Na
+
/K
+
ATPase [16]. Molecular distance
measurements after specific labeling of the high and low
affinity ATP sites with these fluorescent probes gave
evidence for the existe nce of a n (ab)
2
dimeric structure
[17]. The finding of full-site, half-site and quarter-site
phosphorylation and reactivities, however, l ed Taniguchi
et al. [12] and Froehlich et al.[18]topostulatetheexistence
of a functional (ab)
4
tetrameric structure of N a
+
/K
+
ATPase.
Correspondence to W. Schoner, Institute of Biochemistry and Endo-
crinology, Justus-Liebig-University Giessen, Frankfurter Str. 100,
D-35392 Giessen, Germany. Fax: +49 641 9938179,
Tel.: +49 641 9938170,

E-mail:
Abbreviations: DANSyl-ATP, 2¢(3¢)-O-(6-N¢,N¢-dimethyl-
aminonaphthalenesulfonyl)ATP; FITC, fluorescein isothiocyanate;
GST, glutathione S-transferase; N-domain, nucleotide domain;
P-domain, phosphorylation domain; pNPP, para-nitrophenylphos-
phate; TNP-ATP, 2¢-3¢-O-(trinitrophenyl) adenosine 5¢-triphosphate;
Tyr-P, O-phospho-
L
-tyrosine.
Enzyme:Na
+
/K
+
exchanging ATPase (EC 3.6.3.9).
Note: This work is part of the dissertatio n of R.K. at Justus-Liebig-
University Giessen, Germany.
(Received 27 May 2004, revised 27 July 2004,
accepted 10 August 2004)
Eur. J. Biochem. 271, 3923–3936 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04330.x
When the three-dimensional structure of the Ca
2+
ATPase pump o f sarcoplasmic reticulum became available
[19], the possibility arose to deduce, by restraint-based
comparative modeling, an analogous three-dimensional
structure of the ATP-binding domain(s) of the H
4
-H
5
loop
of Na

+
/K
+
ATPase [20]. The overall structure for the loop
between L354 and L 773 excellently predicted the r eal
structure which was obtained much later by crystallization
and NMR spectroscopy [21,22]. Some deviations from
Hakanson’s crystal structure of a much shorter nucleotide
loop (R378–D586) were noted, however. Hence, some
corrections were recently performed to interpret v ariations
in the location of ATP and 2¢-3¢-O-(trinitrophenyl) adeno-
sine 5¢-triphosphate, trisodium salt (TNP-ATP) binding
within the N-domain (L354–I604) [23]. In silico docking of
ATP as well as NMR studies demonstrated that the H
4
-H
5
loop consisting of the N- and phosphorylation (P)-domains
contains a s ingle ATP site only on the N-domain [20].
Nevertheless, affinity labeling by [
32
P]8-azido-ADP[aP] of
FITC-inactivated and membrane-embedded Na
+
/K
+
ATPase revealed that an amino a cid sequence residing on
the P-domain C-terminally of K736 is involved in ADP
recognition [24,25]. K736 of the a subunit has formerly been
shown to participate in ATP hydrolysis [26,27]. So far it is

unclear whether the P-domain’s ADP site may be used when
ADP in the overall reaction leaves the active site, i.e. after a
bending of the N- toward the P-domain connected with the
phosphorylation of D369, or whether the a subunit may
contain two ATP sites. I n fact, structural analysis of Ca
2+
ATPase crystals revealed the existence of at least three
different conformers [19,28,29].
It is generally assumed that the activity of a K
+
activated
phosphatase resides in or close to the ATP binding site and
that upon blockade of the A TP site (for instance by
modification with FITC [13]), the remaining K
+
phospha-
tase reflects a property of the ATP site [12,30–33]. The K
+
activated phosphatase which is inhibited by cardiac glyco-
sides presumably reflects the K
+
activated step of the
hydrolysis of the D369–phosphointermediate in the overall
reaction cycle. Acylphosphates and p-nitrophenylphosphate
(pNPP) may phosphorylate the enzyme protein [10–12,33].
With the demonstration that the isolated H
4
-H
5
loop

expressed in Escherichia coli retains both TNP-ATP binding
[23,34–36] and phosphatase activity [36], the possibility
arose to localize both activities within this loop and to study
their properties. This paper localizes by truncation, single
site mutation and in silico docking experiments, the position
of the binding site for ATP at the front side of the N-domain
between I390 and L576 and reveals the separate existence
of a p-nitrophenylphosphatase at the rear site o f the
N-domain. Our studies gave no indication of a s econd
ATP binding site in the self-forming conformer of the
isolated H
4
-H
5
loop.
Experimental procedures
All chemicals were of the highest purity available and were
obtained from Applichem (Darmstadt, Germany), Bio-Rad
(Munich, Germany), B oehringer-Mannheim ( Mannheim,
Germany),E.Merck(Darmstadt,Germany),Sigma-
Aldrich (Taufkirchen, Germany) Molecular Probes
(Eugene, OR, USA) or Carl Roth (Karlsruhe, Germany).
Pfu-polymerase was from Stratagene (La Jolla, CA, USA)
and the restriction endonucleases BamHI and EcoRI were
from Promega ( Mannheim, Germany). The pGEX-2T
expression vector was from Amersham Biosciences (Frei-
burg, Germany). DNA miniprep and DNA gel extraction
kits were from peqLab (Erlangen, Germany) and Qiagen
(Hilden, Germany). Supercompetent E. coli XL1 b lue cells
were bought from Stratagene. BL21DE3 cells were a

generous gift from J. Naprstek (Charles University, Prague,
Czech Republic). DNA sequencing was performed on an
ABI Prism automated sequencer at the facility of the
Academy of Sciences of the Czech Republic. Calculation
and presentation of the data were performed with
GRAPH-
PAD PRISM
3.0 (GraphPad Software, San Diego, CA, USA).
Protein–protein amino acid sequence comparisons were
performed by
BLAST
analysis ( />BLAST/Blast.cgi) using the SwissProt Data B ank.
Enzyme and assays
Na
+
/K
+
ATPase from pig kidney with a specific activity
of 17 UÆmg protein
)1
[37] was quantitated by a coupled
spectrophotometric assay [38]. One enzyme unit (U) is
defined as the amount of enzyme hydrolyzing 1 lmol ATP
per minute at 37 °C. Protein concentration was d etermined
by Lowry’s procedure for the membrane-bound Na
+
/K
+
ATPase [39] but by the method of Bradford [40] for the
H

4
-H
5
loop–glutathione S-transferase (GST) fusion protein
and its truncation products. K
+
activated p-nitrophenyl-
phosphatase as a partial activity of Na
+
/K
+
ATPase was
measured as described previously [17]. S tatistical analysis of
the comparison of the phosphatase activities in truncated
H
4
-H
5
loop was carried out with Student’s t-test.
Construction and purification of H
4
-H
5
loop–GST fusion
proteins
The part of the DNA sequence of the a subunit of mouse
brain Na
+
/K
+

ATPase encoding for the large cytoplasmic
loop (L354–I777) was amplified by PCR. The purified PCR
product was digested with BamHI an d EcoRI. The expres-
sion vector pGEX-2T (Amersham Pharmacia) was opened
with the restriction endonucleases BglII and EcoRI. The
insert was ligated into the multiple cloning site of the doubly
digested vector downstream of the GST coding sequence.
With the pGEX-2T–H
4
H
5
construct, supercompetent
E. coli XL1 blue cells (Stratagene) or BL21DE3 cells were
transformed. Starting with this vector, l oops of different
lengths were designed by the insertion of stop codons into
the sequence at the positions of K605, R589, C577, G542
and K528, respectively. The N-terminal shortened construct
I390–S601 was m ade by subcloning the c orresponding
DNA sequence into the multiple cloning site of an empty
pGEX-2T v ector between Bam HI and EcoRI sites as
described above.
The following primers were used for the amplification of
different constructs, with the relevant site underlined in each
case: L 354–I777 sense with BglII site: 5¢-C GT
AGATCT
CTGGAAGCTGTGGAGACC-3¢;antisensewithEcoRI
site: 5¢-AT
GAATTCCAATGTTACTTGTTAGGGT-3¢;
L354–I604 sense with stop codon: 5¢-CAGCGCTGG
GATT

TAGGTCATCATGGTC-3¢; antisense with stop
3924 R. Krumscheid et al.(Eur. J. Biochem. 271) Ó FEBS 2004
codon: 5¢-CTCCTGTGACCATGATGACCTAAATCCC
AGC-3¢; I390–S601 sense with BglII site: 5¢-GC GT
AGA
TCTATCCATGAAGCTGACACCACAG-3¢;antisense
with EcoRI restriction site: 5¢-AT
GAATTCGCGCTGCG
GCATTTGCCCACAGC-3¢; L354–P588* sense with stop
codon: 5¢-ATTGACCCTCCT
TGAGCTGCTGTCCCCG
ATGCTGTG-3¢; L354–L576* sense with stop codon:
5¢-CCCGTGGATAACCTC
TGATTCGTGGGTCTTAT
CTCC-3¢; L354–L541* sense with stop codon: 5¢-GGCC
TTGGA
TAGCGTGTGCTAGGTTTCTGCCACCTC-3¢;
L354–L527* sense with stop c odon: 5¢-C CCCTGGACGA
AGAGCTG
TAAGACGCCTTTCAGAATGCC-3¢;the
* means that antisense primers of the C-terminally shor-
tened constructs were usually complementary. The primer
sequence of the N398D construct was GCTGACACCA
CAGAG
GATCAGAGTGGGGTCTCC and that of the
D369A construct C CACCATCTGCTCC
GCCAAGACT
GGAACTCTGAC. The underlined nucleotides encode the
mutated amino acid.
Expression and purification of the GST fusion proteins

was performed according to Kubala et al. [41]. The purity of
the expressed protein was controlled by 12% SDS/PAGE
and its concentration w as determined by the method of
Bradford [40] using diluted protein standard (80 gÆL
)1
)from
Sigma.
All experiments described b elow were performed with
protein samples that had been dialyzed extensively over-
nightat4°C against an excess of 20 m
M
Tris/HCl, pH 7.8,
with one buffer change.
Phosphatase assay of the H
4
-H
5
loop fusion proteins
The a ssay was performed in variation of t he procedure
described by Tran & Farley [36]: GST fusion proteins
(about 50 lg per sample) w ere i ncubated in a buffer
containing 64 m
M
Tris/HCl, 3 .2 m
M
MgCl
2
,8m
M
KCl

and 0.8 m
M
Na
4
EDTA, pH 7.4, at 37 °Cwithincreasing
concentrations of pNPP (0–2 m
M
) i n a total volume of
1 mL. The r eaction was stopped after 24–48 h by
addition of 3
M
NaOH. Proteins were sedimented and
the absorption of the supernatant was monitored at
405 nm. Background (hydrolysis of pNPP under the
same conditions in the absence of protein) was
substracted. The velocity of substrate cleavage was
calculated assuming a molar absorption coefficient of
18 500 LÆmol
)1
Æcm
)1
. D ata were fitted to the Michaelis–
Menten equation.
Test for protein tyrosine phosphatase activity
The assay was performed using the EnzCheck Phosphate
Assay Kit (Molecular Probes, Eugene, O R, USA). T he
GST fusion proteins L354–I777 and L354–I604 were
tested for their ability to release phosphate from
O-phospho-
L

-tyrosine (Tyr-P). The proteins were incuba-
ted under the condition s of the phosphatase assay w ith
5m
M
Tyr-P. After 22–42 h of reaction time, the
following reagents were added to 500 lLofeach
reaction mix: 345 lLH
2
O, 50 lL20· reaction buffer,
100 lL 2-amino-6-mercapto-7-methyl-purine r iboside,
5 lL purine nucleoside phosphatase. The samples were
mixed and incubated for 30 min at room temperature.
Detection of the released phosphate was recorded at
360 nm. Cont rols were withou t prote in/Tyr-P and w ere
run in p arallel.
Determination of TNP-ATP binding to the fusion proteins
Steady-state fluorescence of T NP-ATP was measured in
20 m
M
Tris/HCl, p H 7.8, a t 37 °CusingaPerkinElmer
LS50B fluorometer. H
4
-H
5
–GST fusion proteins (2 mL;
1 l
M
GST fusion protein in a 1 · 1 cm quartz cuvette) were
titrated with increasing concentrations of TNP-ATP. Exci-
tation and emission wavelengths were recorded at 405 nm

and 545 nm, respectively, after 3 min of incubation at 37 °C
in the dark and gentle stirring. TNP-ATP binding to the
protein was detected as an increase of fluorescence intensity
in the p resence of p rotein compared to the fluorescence
intensity in its absence [23,42].
Determination of eosin binding to the fusion proteins
Interaction of eosin Y with GST fusion proteins was studied
in similarity to Skou & Esman [43] in 20 m
M
Tris/HCl,
pH 7.8, at 37 °C. Excitation (480–530 nm with k
Emm
¼
538 nm) and emission (530–580 nm with k
Exc
¼ 518 nm)
spectra in the presence and absence of 1 l
M
or 10 l
M
(L354–P588)–GST fusion protein were recorded on a
Hitachi F-3000 Fluorescence Spectrophotometer with
5 nm bandpass. Steady-state fluorescence studies were
performed with the (L354–I777)–GST fusion protein in
thesamebufferat37°C on a PerkinElmer LS50B
Luminescence Spectrometer exciting the probe at 518 n m
and recording the emitted fluorescence at 530 nm (5 nm
band passes each) and using an emission filter of 530 nm.
The following ligands were tested with respect to their
influence on the steady-state fluorescence of 100 n

M
eosin Y
in 20 m
M
Tris/HCl, pH 7.8, in the presence of the H
4
-H
5
loop: 10 m
M
Na
+
,5m
M
Mg
2+
,5m
M
PO
4
3–
,1.5m
M
and
3m
M
ATP.
Calculation of the dissociation constants for TNP-ATP
The signal of buffer and protein (if present) was collected
before the addition of TNP-ATP, and this value was

subtracted from all further raw data as a background.
Volume corrections were applied and background values of
TNP-ATP fluorescence in the absence of GST f usion
proteins were subtracted. Fluorescence intensity was nor-
malized so that a fluorescence of 1 l
M
TNP-ATP (i.e. in the
absence of protein) w as equal to unity. The dependence of
fluorescence intensity on the concentration of TNP-ATP
was fi tted to Eqn (1) [44], describing a model w ith one
binding site per protein molecule:
F ¼½Pþ
1
2
ðc À 1Þ
½Pþ½EþK
d
À
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ð½Pþ½EþK
d
Þ
2
À 4½P½E
q

ð1Þ
or to Eqn (2) [44], describing a model with n identical,
noninteracting, noncooperative binding sites per protein
molecule:

Ó FEBS 2004 Mapping of ATP and pNPP binding sites of the Na pump (Eur. J. Biochem. 271) 3925
F ¼½Pþ
1
2
ðc À 1Þ
½Pþn½EþK
d
À
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ð½Pþn ½EþK
d
Þ
2
À 4n½P½E
q

ð2Þ
where F is the normalized fluorescence, [P] is the concen-
tration of TNP -ATP, [E] is the concentration of the enzyme,
c is the enhancement o f fluorescence intensity of the bound
probe relative to the free probe, and K
d
is the dissociation
constant.
The value of the quantum yield enhancement factor c was
assessed according to [44] and determined as 7 ± 0.7 for the
H
4
-H
5

loop [41]. A ll parameters except K
d
were kept
constant during the fitting procedure. Data are presented as
mean ± SEM from the indicated number of independent
measurements.
Modification of the ATP binding site by FITC labeling
for phosphatase studies
Na
+
/K
+
ATPase from pig kidney (6 U; 300 lg; 2 l
M
)was
incubated in a t otal volume of 1 mL for 30 min in the dark
at room temperature in a solution of 40 m
M
Tris/HCl, pH
9, and 10 l
M
FITC. Excess fluorophore was removed by
sedimentation of the protein a t 100 000 g in an ultracentri-
fuge. Modification of 5 l
M
of the (L354–I604)–GST fusion
protein proceeded for 2 h in 40 m
M
Tris/HCl,pH9,inthe
presence of 10 l

M
FITC. Free fluorophore was removed by
dialysis overnight a t 4 °C against a large volume of 40 m
M
Tris/HCl, pH 7.4. Binding of FITC was determined as the
molar ratio of FITC bound per H
4
-H
5
loop or per the a
subunit of Na
+
/K
+
ATPase [17]. K
+
activated phospha-
tase activity of the A TPase as well a s the phosphatase
activity of the H
4
-H
5
loop protein were tested as described
above [14,17].
Labeling and purification of loop protein without
the GST tag for fluorescence anisotropy studies
GST fusion protein L354–P588 (15 l
M
)in20 m
M

Tris/HCl,
pH 9, was labeled for 30 min with 30 l
M
FITC in the dark
at room temperature. Residual free FITC was removed by
dialysis over night against a large excess of 50 m
M
Tris/HCl,
150 m
M
NaCl, 2.5 m
M
CaCl
2
, pH 7.8. The GST tag was split
off by 10 U of human thrombin per mg of GST fusion
protein for 1 h at room temperature with gentle shaking. The
GST protein was removed by incubation of the mixture with
1 mL of pre-equilibrated glutathione Sepharose (see above).
This procedure was repeated once more. Finally, thrombin
and buffer c omponents w ere r emoved by size exclusion
chromatography on a 3 mL Sephadex G-25 column pre-
equilibrated with 20 m
M
Tris/HCl, pH 7.8. The concentra-
tion of the FITC labeled loop was 145 lgÆmL
)1
(2.88 l
M
),

the molar ratio of FITC bound to peptide was 1.2.
Molecular modeling
Molecular modeling of the H
4
-H
5
cytoplasmic loop of the
a subunit of Na
+
/K
+
ATPase ranging from L354 to
L773 has been reported previously [20]. Models of the
cytoplasmic loop of mouse brain Na
+
/K
+
ATPase from
L354–I604, L35 4–L541, L354–L527 a nd I390–S601 were
generated in parallel to Ca
2+
ATPase (PDB code 1EUL)
[19] with the
MODELLER
6 package [45]. The tertiary structure
models were checked with
PROCHECK
[46], showing g-factors
in the same range as reported in [41] for the pig kidney loop.
A model of the N-domain of mouse brain a

1
subunit
Na
+
/K
+
ATPase (R378–D586) was generated by analogy
to the crystal structure of the corresponding sequence of
porcine a
2
sodium pump [21,47]. The latter, recently
published structure lacks three parts of 6, 10 and 6 amino
acid residues that exist in the mouse brain a
1
subunit.
Hence, the three-dimensional structure of these three
peptides was additionally modeled according to the proce-
dure published previously for the H
4
-H
5
loop of pig kidney
Na
+
/K
+
ATPase [20]. The primary structure of the mouse
brain Na
+
/K

+
ATPase from R378 to D586 was aligned
with the template sequences by
CLUSTALX
[48]. The three-
dimensional model constituted by all nonhydrogen atoms
was built and examined by the
MODELLER
6 package [45,48].
The tertiary structure model was checked with
PROCHECK
[46].
Ligand docking
The crystal structure of pNPP was extracted from the PDB
coordinates file, 1D1Q [49], deposited in the Protein Data
Bank (). Hydrogens were added using
the
BIOPOLYMER
module included in
INSIGHT II
(Accelrys
Inc.,SanDiego,CA,USA).
Docking of ATP, TNP-ATP and pNPP was explored
with
AUTODOCK
[50]. To complete modeling of the
truncated peptides, energy minimization and docking
procedure was performed using exactly the parameters
and methods published for pig kidney Na
+

/K
+
ATPase
[20,23]. Several dynamics runs were set up for a canonical
ensemble. One dynamics run was a single i nterval of
120 ps at 3 00 K, and 343 K, respectively, with a femto-
second time step result being r ecorded every 25 fs. The
shake technique was applied to all bonds. Force field
parameters were the same as for the minimization. FITC
was connected to K501 via a covalent bond using the
BUILDER
module included in
INSIGHT II
and its position in
the binding site was optimized.
Results
Effects of truncation of the cytoplasmic H
4
-H
5
loop of the
a subunit of Na
+
/K
+
ATPase on TNP-ATP binding and
p
-nitrophenylphosphatase activity
Molecular modeling of the H
4

-H
5
loop according to the
E1-Ca
2+
ATPase [20] gives almost identical results for the
N- and P-domains as with its crystal structure or NMR
analysis [21,22]. In silico docking of ATP and TNP-ATP to
the H
4
-H
5
loop showed a single ATP binding site only [41]
(Fig. 1A,B). In the active site residing between I390 and
L576 (Table 1), eight amino acids interact with ATP [41].
To ensure that docking experiments in fact reflect properties
of the loop in solution, we analyzed TNP-ATP equilibrium
binding to the (L354–I777)–GST fusion protein that
contains both the N- and P-domains. Titration in fact
revealed that TNP-ATP binds to a single site only, as fitting
3926 R. Krumscheid et al.(Eur. J. Biochem. 271) Ó FEBS 2004
TNP-
ATP
D369
N-Domain
P-Domain
A
B
C
L354

I604
L354
L527
ATP
S477
F475
Q482
E446
L354
E505
K501
K480
L773
Fig. 1. C-Terminal truncation of the H
4
-H
5
loop leads to loss of TNP-ATP binding due to unfolding of the N-domain as revealed by molecular
modelling. Molecular modeling was performed as described previously [20]. (A) The complete H
4
-H
5
loop starting at L354 and ending at L773
contains the nucleotide (N)-domain interac ting with TNP-ATP and th e phosphorylation (P)-d omain (D369 shown in blue). The amino acid
sequence ALLK known to interact with a nkyrin [60] is colored in green. (B) The size of the isolated H
4
-H
5
loops shortened by the C-terminal part of
the P-domain to L354–I604 is without effect on TNP-ATP binding. (C) The stability of the N-domain and its ability to bind TNP-ATP with high

affinity is lost, however, when the sequence C-terminally of L527 is removed, although all the amino acids known to interact directly with ATP
[41,60–63], and shown in purple are still present (E446, Q482, F475, S477, K480, K501, E505). The mobility of the structure in the residual
N-domain of L354–L527 forming the ATP binding site is indicated by red arrows. Each of the displayed proteins contains an unaltered Mg
2+
dependent p-nitrophenylphosphatase activity.
Table 1. TNP-ATP binding to and p-nitrophenylphosphatase of H
4
-H
5
loop–GST fusion proteins of different lengths. Theincreaseoftheintensityof
fluorescence by TNP-ATP binding was recorded using various H
4
-H
5
loop–GST fusion proteins of different lengths as described in Experimental
procedures. The K
d
values were calculated using eqn (1). The number of binding sites for ATP/loop was 1 for all investigated proteins. The activity
of p-nitrophenylphosphatase was measured at 37 °C as d escribed in Experimental procedures. Mean values ± SEM are given from two to six
independent experimen ts.
Amino acid sequence
or mutation
Length/amino acids
(without GST)
TNP-ATP binding
K
d
(l
M
)

Mg
2+
activated phosphatase
K
m
(l
M
) V
max
(nmolÆh
)1
Æmg
)1
)
L354–L777 324 3.55 ± 0.35 0.76 ± 0.05 10.35 ± 0.73
L354–I604 251 3.30 ± 0.06 0.83 ± 0.07 11.28 ± 0.72
I390–S601 212 3.50 ± 0.07 0.57 ± 0.08 5.22 ± 1.08
L354–P588 235 2.95 ± 0.05 0.88 ± 0.03 11.36 ± 0.48
L354–L576 222 3.60 ± 0.07 0.83 ± 0.08 11.42 ± 2.42
L354–L541 188 4.73 ± 0.19 0.88 ± 0.22 9.18 ± 1.00
L354–L527 174 10.05 ± 0.95 1.10 ± 0.18 8.75 ± 1.30
N398D (L354–I604) 251 3.30 ± 0.2 0.93 ± 0.10 5.65 ± 0.53
D369A (L354–I604) 251 3.50 ± 0.50 1.17 ± 0.15 8.96 ± 0.24
Ó FEBS 2004 Mapping of ATP and pNPP binding sites of the Na pump (Eur. J. Biochem. 271) 3927
of the fluorescence enhancement to a single site nucleotide
binding model (Fig. 2), gave no indication for a second site.
TNP-ATP b inding was suppressed b y the presence of ATP
and ADP but not by AMP, as previously reported [34,35]
(data not shown).
A further means to s earch for a second ATP and

phosphatase binding site is to prepare shorter loop
constructs and t o loo k for their abilities to b ind T NP-
ATP and to hydrolyze pNPP. Consequently, a number of
GST fusion proteins starting at L354 and ending at varying
C-terminal ends were expressed and purified. The constructs
without the C-terminal part o f t he P-domain showed
unaltered K
d
values o f t he loop–TNP-ATP complexes
(oscillating around a mean value of 3.35 l
M
)aswellas
unaltered properties of a Mg
2+
dependent phosphatase
activity, as long as C-terminal shortening did not exceed
L576 (Table 1). The TNP-ATP binding properties c hanged
drastically, however, when C-terminal shortening down to
L527 took away parts of the N-domain that a pparently
stabilized its backbone (Fig. 1C). We observed an approxi-
mately 40% increase of the dissociation constant K
d
for the
shortened construct, L354–L541 (K
d
¼ 4.73 l
M
), but a
sharp significant increase of the K
d

for TNP-ATP for
the shortest construct, L354–L527 (200%, K
d
¼ 10 .05 l
M
;
Table 1 ). We should add that shorter constructs could not
be purified because they showed an increasing tendency to
precipitate in solution. This shortest construct L354–L527
still contained all amino acids known to be necessary to bind
ATP (Fig. 1C) [46].
Amino terminal shortening of the loop protein was also
tested. We prepared the construct I390–S601 which lacks
the phosphorylation site at D369. This construct showed a
single TNP-ATP binding site as well. The protein had the
same TNP-ATP binding properties as the longest protein
L354–I777 with K
d
¼ 3.5 0 l
M
(Table 1). Interestingly, its
Mg
2+
activated phosphatase activity was more than 50%
reduced as compared to the corresponding construct L354–
I604 containing the N-terminal part o f the P-domain.
Studies on the structural stability of the N-domain by
FITC anisotropy decay and eosin fluorescence
Overall Na
+

/K
+
ATPase activity may start with ATP
binding to the N-domain. This process may eventually
induce its bending towards the P-domain and thereby
explain the amino acid labeling of the P-domain found with
8-N
3
-TNP-ADP [25]. The same consideration may apply to
thelabelingofP668by4-N
3
-2-NO
2
-phenylphosphate [51].
It was therefore of interest to learn more on the rigidity of
the N-domain. This issue was investigated by FITC
fluorescence anisotropy decay and lifetime measurements
as well as by steady-state eosin fluorescence stu dies.
FITC fluorescence anisotropy decay measurement. FITC-
labeled L354–P588 loop protein with a molar ratio of
fluorophore/protein o f 1.2 was prepared a s d escribed in
Experimental procedu res. Its steady-state fluorescence
anisotropy of r ¼ 0.25 was quite high for a soluble protein,
indicating a low flexibility of the N-domain. This value is,
however, significantly lower than the r ¼ 0.34 of the FITC-
labeled and membrane-embedded Na
+
/K
+
ATPase [52].

Additionally, to have a closer look to the rigidity of the
FITC-labeled L354–P588 H
4
-H
5
loop, the lifetime of the
excited state and the anisotropy decay of the labeled protein
were determined using a phase domain fluorometer with
modulation f requencies f rom 10 MHz to 200 MHz. We
observed a two-component fluorescence intensity decay
with the major lifetime component s
1
¼ 3.5 ns (f
1
¼ 0.77)
and the minor component s
2
¼ 1.7 ns (f
2
¼ 0.23). The
average lifetime of the excite d state was determined as s ¼
3.1 ns.
The anisotropy decay of FITC-labeled L354–P588 H
4
-H
5
loop was determined in L-format over the range of
modulation frequencies from 10 M Hz to 200 MHz. A
two-component decay with a longer component of q
1

¼
11.3 ns and a shorter component of q
2
¼ 1.2 ns, seemed to
satisfactorily fit the collected data. The shorter component is
short enough to b e ascribed to t he wobbling o f the
fluorophore around its binding site. The longer component,
on the other hand, is long enough to reflect the motion of
the whole FITC-labeled H
4
-H
5
loop and not only segmental
motions. C onsequently, we have to co nclude that the
N-domain containing the ATP-binding site is rigid without
any flexible segments.
Eosin binding to the H
4
-H
5
loop–GST fusion pro-
teins. Eosin Y is a well studied fluorescence label competing
with ATP for its binding site in the membrane-embedded
enzyme. It has been used to demonstrate ATP competition
as well as Mg
2+
and K
+
induced conformational changes
in Na

+
/K
+
ATPase [43,53,54]. The largest construct, the
(L354–I777)–GST fusion protein, and the C-terminally
shortened construct L354–P588 were used for a comparat-
ive study. I n contrast to results reported for the membrane-
embedded Na
+
/K
+
ATPase [43,54], we observed neither a
change of the excitation nor of the emission fluorescence
spectra in the presence of any of these H
4
-H
5
loop–GST
Fig. 2. Binding of TNP-ATP to the (L354–I777)–GST fusion protein,
and fit of the data to equations for 1 or 2 TNP-ATP binding sites. The
(L354–I777)–GST fusion protein (1.6 l
M
) was titrated with TNP-ATP
in 50 m
M
Tris/ H Cl , pH 7 .5 at 3 7 °C. Excitation and emission wave-
lengths were 462 nm and 527 nm, respectively. Regression analysis
according to the Eqns (1) and (2) (solid line: Eqn (1); broken line:
Eqn (2); also Table 1) demonstrates that the e quation describing the
properties of a single TNP-ATP binding site gives the best fit.

3928 R. Krumscheid et al.(Eur. J. Biochem. 271) Ó FEBS 2004
fusion proteins. No detectable i nfluence of Na
+
,Mg
2+
,
PO
4
3–
or ATP on the eosin Y steady-state fluorescence was
seen in the presence of 1 l
M
GST fusion protein L354–I777
(data not shown). This is rather surprising as Costa et al.
reportedonaneosininterferencewithMgATPinthedimer
formation of a H
4
-H
5
loop protein of Na
+
/K
+
ATPase
[55]. In conclusion, neither method revealed any indication
of a conformational change of the N-domain upon ligand
binding in the investigated GST fusion protein. It is quite
evident that t he N-domain of t he isolated loop forms a
rigid structure, unless essential parts of the backbone are
removed (Fig. 1C).

Characterization of the three-dimensional structure of
the complete and truncated H
4
-H
5
loops by molecular
modeling
The interpretation o f the above r eported data on the
truncation of the H
4
-H
5
loop is considerably facilitated by
the availability of a molecular model [20]. Molecular
modeling of the truncated H
4
-H
5
loop revealed t hat a big
part of the N-domain can be removed without any loss in
TNP-ATP binding properties (compare Table 1 with
Fig. 1A–C). The increase i n K
d
value o f the TNP-A TP
protein complex by the extreme C-terminal shortening to
the L354–L527 construct could be described by dynamic
and energy minimization runs to be the result o f an
increased mobility of parts of the loop structure (Fig. 1C).
The location of FITC within the ATP site obtained either
by in silico docking to the previously described full H

4
-H
5
loop model [20] or to the N-domain model according to
Hakansson’s crystal structure [21,23] revealed that both
models shows an ionic interaction of the carboxyl-group of
E446 with the e-amino group of the modified K501 (Fig. 3).
Both models also show that F475 interacts with the
aromatic moiety of the FITC label, similarly to ATP and
TNP-ATP [23,41,56]. There is, however, a distinct difference
with respect to the location o f F548: the model c reated
according to H akansson’s crystal s tructure (R378–D586)
[23] (Fig. 3B) shows F548 buried under the surface of the
ATP binding pocket, while the model for t he bigger H
4
-H
5
loop (L354–L773) (Fig. 3A) allowed aromatic interactions
with substrates and inhibitors.
Studies on the localization of a
p
-nitrophenyl-
phosphatase activity within H
4
-H
5
loop–GST fusion
proteins
Consistent with a previous report [36], shortening of the
C-terminal part of the H

4
-H
5
loop down to amino acid
number 600 revealed no change in phosphatase activity
(Table 1). This may mean that the phosphatase is located on
a part of the N-domain. Because a transfer of the phosphate
group of pNPP to the protein has been reported for the
membrane-embedded enzyme [11,33], a possible participa-
tion of the phosphorylation s ite D369 in the isolated H
4
-H
5
loop’s p-nitrophenylphosphatase activity was tested. Muta-
tion of D369 to A had no significant effect on the V
max
of
substrate hydrolysis (Fig. 4). We therefore conclude that the
isolated loop does not form a phosphointermediate during
catalysis. Interestingly, when the amino terminal part of the
P-domain was deleted, the resulting (I390–S601)–GST
fusion protein showed significantly lower V
max
and K
m
values for pNPP (Table 1, F ig. 4). Because the Mg
2+
dependent phosphatase of the H
4
-H

5
loop shows a very low
turnover rate, a ttempts w ere mad e to build up a more
Fig. 3. Recognition of FITC by amino acids forming the ATP binding site of the N-domain. (A) Model of the whole loop in analogy to the E1-Ca
2+
ATPase structure (N- and P-domains, L354–L773) [20]. Covalent coupling of FITC to K501 is accomplished by a hydrophobic interaction of the
benzoyl-group within FITC with F548. The tricyclic residue of FITC cover s most of the space of the nucleotide binding site. (B) Model based on
the N-domain c rystal structure of a
2
Na
+
/K
+
ATPase (N-domain only, R378–D586) [23]. F548 is buried under the surface of the pro tein and
therefore not able to interact with ligands in the nucleotide binding pocket. Both models imply an ionic interaction of the carboxyl-group of E446
with the e-amino-group of the modified K501. The covalent bound label seems to be stabilized by interaction with the aromatic side chain of F475.
Ó FEBS 2004 Mapping of ATP and pNPP binding sites of the Na pump (Eur. J. Biochem. 271) 3929
sensitive assay using 3-O-methyl-fluorescein phosphate;
this substrate has formerly been shown to be hydrolyzed
K
+
-dependently by Na
+
/K
+
ATPase [30,57]. Unfortu-
nately, however, it was impossible to use this substrate to
investigate the phosphatase activity of the isolated lo op due
to its high rate of autohydrolysis. Phosphotyrosine was also
tested but not hydrolyzed by the (L354–I777)– and (L354–

I604)–GST fusion proteins.
Surprisingly, FITC-labeling o f the loop protein i n the
H
4
-H
5
loop had only a small effect on Mg
2+
dependent
phosphatase activity (Table 2). The observed effect is in
the same range as for the K
+
activated, Mg
2+
depend-
ent phosphatase activity in the membrane-embedded full
enzyme (Table 2). Hence, it seemed possible that a pNPP
binding site might exist separately from the ATP site.
Tran & Farley had reported that N398 is labeled by
radioactive 4-azido-2-nitrophenylphosphate and that this
labeling l eads to an inactivation o f Na
+
/K
+
ATPase
[51]. pNPP docking experiments to the H
4
-H
5
loop

indicated that this s ubstrate may interact with the
nucleotide binding site as well as with a strand of three
b sheets at the rear surface (pNPP site, Fig. 5). A closer
look at these areas revealed that within the ATP site, the
aromatic ring of pNPP may interact with t he phenyl
residues of F475 and, if accessible, also with F548
(Fig. 6 A,C). The NO
2
group of pNPP, however, m ay
interact with Q482 and K 501 of the nucleotide binding
site in both models (Fig. 6A,C).
The calculated docking energies were )6.7 kcalÆmol
)1
for
the structure of L354–L773 derived from E1-Ca
2+
ATPase
(Fig. 6 A) and )6.4 kcalÆmol
)1
for the N-domain analogy
model according to Hakansson (R378–D586) [21] (Fig. 6C).
These estimations of interaction energies neglect solvation
and desolvation. The phosphate group of pNPP is about
2.8 nm from the phosphorylation site D369.
The location of the putative pNPP binding sites on the
rear part of the loop differed significantly depending on
the presence or absence of the N-terminal part of the P-
domain (Fig. 5B,D). The calculated interaction energies
for both models were )7.5 (Fig. 5B) and )6.8 kcalÆmol
)1

(Fig. 5D), respectively. In the Ca
2+
ATPase derived
model [20], a hydrophobic environment was formed by
M384, L414, W 411, which may stabilize the substrate’s
phenyl ring from both sides (Fig. 4, Table 1). The NO
2
group of pNPP seems to interact w ith N 398, while the
phosphate group may be stabilized by interaction with
S400 and S408 (Fig. 6B). Docking to a model based on
Hakansson’s crystal structure [21,23] missing the N-
terminal part of the P-domain (R378–D586), however,
showed a d ifferent pNPP binding site 16 A
˚
away f ro m
N398 with an estimated 10% lower interaction energy as
compared to the full loop (Fig. 5B,D). I n this case, the
NO
2
group seems to point to the direction of S408, while
the phosphate group may lie between S401 and Q389.
The only hydrophobic interaction of the phenyl ring i n
this model is achieved by H517.
In the Ca
2+
ATPase-derived model (Fig. 5A), the phos-
phate group of pNPP is 3.2 nm from the phosphorylation
Fig. 4. Truncation of the residual P-domain, mutation of the phosphorylation site D369, and N398 as part of the putative phosphatase site. Effects on
V
max

and K
m
of Mg
2+
dependent p-nitrophenylphosphatase activity. The effect o f single site mutation or N-terminal truncation of the (L354–I604)–
GST fusion proteins of the H
4
-H
5
loop on the activities and properties of the p-nitrophenylphosphatase activity was studied. The properties of the
control refer to th e mean values o f six different constructs of carbox y terminally truncated H
4
-H
5
loop starting at L354 and ending carboxy
terminally between L527 and I777 (Fig. 3). The V
max
and K
m
values of the Mg
2+
dependent p-nitrophenylphosphatase activity as a function of the
phosphorylation site D369, the presence or loss of the residual P-domain (L354–Q389), and the function of N398 in the putative phospatase site are
shown. The significance of the difference s in activity was evaluated by two-tailed student’s t-test as: * P <0.05, ** P < 0.01, *** P <0.001.
Table 2. Effect of FITC-labeling on the phosphatase activities of Na
+
/
K
+
ATPaseandaH

4
-H
5
loop–GST fusion protein. Labeling of pig
kidney Na
+
/K
+
ATPase as well as the purified L354–I604 loop pro-
tein was performed with 10 l
M
FITC at pH 9 fo r 30 min and the
labeled protein was handled as describedinExperimental procedures.
Phosphatase activity of the L354–I604loopproteinwastestedat5 m
M
pNPP. NA, not applicable.
Molar binding
ratio of FITC
Phosphatase
activity
Na
+
/K
+
ATPase
(control)
NA 1.54 lmolÆmg
)1
Æmin
)1

(100%)
L354–I604 (control) NA 7.84 nmolÆmg
)1
Æh
)1
(100%)
Na
+
/K
+
ATPase +
FITC
0.8 ± 0.2 1.23 lmolÆmg
)1
Æmin
)1
(80%)
L354–I604 + FITC 0.9 ± 0.1 6.58 nmolÆmg
)1
Æh
)1
(84%)
3930 R. Krumscheid et al.(Eur. J. Biochem. 271) Ó FEBS 2004
site D369 (not shown). To test the hypothesis of an
additional phosphatase site and to locate this site, N398
was mutated to aspartate. The mutation led to an
approximately 50% decreas e of the p-nitrophenylphos-
phatase activity connected wi th a slight decrease in the
affinity (Fi g. 4). TNP-ATP b inding was not affected by
this mutation (Table 1).

Even millimolar concentrations of ATP did not inhibit
the phosphatase activity (data not shown), indicating that
the pNPP site is unable to bind ATP and that within the
isolated H
4
-H
5
loop protein, binding of ATP to the
nucleotide binding site does not lead to a conformational
change of the N-domain, or to an alteration of the pNPP
site.
Fig. 5. p-Nitrophenylphosphate can be docked to the ATP site as well as to a phosphatase site at the rear surface of the N-domain (overview). (A)
Docking of pNPP to the ATP binding site of the model containing N- and P-domains [20]. The amino acids interacting with the adenine ring of
ATP may also interact weekly with p-nitrophenylphosphate (pNPP). (B) pNPP docked to a surface around N398 at the rear site of the N-dom ain
(pNPP site) of the same model. (C) Docking of pNPP to the ATP binding site of the model containing the N-domain only, modeled according to
Hakansson’s crystal structure [21,23]. Comparison of (A) and (C) show little structural difference in the environment of the docked ligand. The final
docking energies w ithout solvation and desolvation effects were estimated as )6.7 kcalÆmol
)1
for (A) and )6.4 kcalÆmol
)1
for structure (C),
respectively. (D) Docking attempts of pNPP to the rear side of the model of the isolated N-domain [23] revealed that this structure does not contain
a binding site aroun d N398 as in (B) (full loop). The in teraction of the substr ate with the N-terminally shortened loop s tructure is lower as compared
to the E1-Ca
2+
ATPase derived structure of the full H
4
-H
5
protein [20] [) 6.8 kcalÆmol

)1
for (B), ) 7.5 kcalÆmol
)1
for (D)].
Ó FEBS 2004 Mapping of ATP and pNPP binding sites of the Na pump (Eur. J. Biochem. 271) 3931
Discussion
Consistent with recently reported data on NMR analysis
[22], TNP-ATP binding studies [34,41] and in silico docking
experiments [20], the above reported data show (Figs 1 and
2, Table 1) that the isolated H
4
-H
5
loop of Na
+
/K
+
ATPase contains a single ATP site only. Former reports on
the existence of two ATP and phosphatase binding sites on
the N- as well on the P-domains [51,58] in the membrane-
embedded sodium pump must reflect the existence of other
protein c onformations [7,8] differing from that of the
isolated H
4
-H
5
loop in solution.
Former studies with pyrene isothiocyanate indicated a
rigid structure of the ATP binding site in the membrane-
embedded sodium pump [59]. Conformational stability of

the N-domain is a lso evident fr om the high steady-state
fluorescence anisotropy o f r ¼ 0.25 for the FITC-labeled
H
4
-H
5
loop and from its long anisotropy decay of q
1
of
11.3 ns favoring the view that the whole loop tumbles in
solution. Additional support for this conclusion comes also
from the fact that the loop does not, in contrast to the
membrane-embedded Na
+
/K
+
ATPase, respond to eosin
Y by fluorescence changes upon addition of ATP, Na
+
or
Mg
2+
[43]. Thus, the N-domain of the isolated H
4
-H
5
loop
is unable to twist down to the P-domain [3]. Such a
conformational change is needed in the membrane embed-
ded Na

+
/K
+
ATPase to enable both, 8-N
3
-TNP-ADP [58]
and 4-azido, 2-nitro-phenylphosphate [51] to label the
P-domain at an amino acid C-terminal of K736 [58] and
at P668 [51]. No indications for binding sites of ATP or
pNPP were detectable in the C-terminal p art of t he P-
domain of the isolated H
4
-H
5
loop (Figs 1 and 2, Table 1).
K
+
activated phosphatase activities in Na
+
/K
+
ATPase
and H
+
/K
+
ATPase supposedly reflect the K
+
activated
step of the h ydrolysis o f an a cyl-phosphointermediate

formed from ATP in both enzymes [12,31]. Kinetic experi-
Fig. 6. A closer look at the ATP and pNPP sites on the N-domain of the H
4
-H
5
loop of Na
+
/K
+
ATPase. The models u sed for (A–D ) are the same a s
in Fig. 5. (A) and (C) This comparison of pNPP binding to the ATP sites of both models shows that in the N- and P-domains con taining model [20]
(A) a hydrophobic interaction of the pNPP’s phenyl resid ue with F475 and F548 exists. The nitro-group of the substrate interacts with K501 and
Q482 in both models. (B) In the model containing N- and P-domains [20], recognition of p NPP by the pNPP site at t he rear su rface of the N -domain
is due the formation of an hydrogen bond of the NO
2
-group of pNPP with the amino group of N398. The hydrophobicity of the binding pocket is
achieved by W411 and L414. The phosphate group seems to form a hydrogen bridge with the OH-group of S408. (D) In the structure based on the
crystal of the a
2
structure of the N-domain [21,23], the N398 does not interact with the substrate. pNPP may bind 16 A
˚
away from this amino acid
residue and with a lower interaction energy as compared to the model in (B).
3932 R. Krumscheid et al.(Eur. J. Biochem. 271) Ó FEBS 2004
ments in the membrane-embedded enzyme could not decide
whether Na
+
/K
+
ATPase and phosphatase sites overlap

[60] or reside on separate sites [51]. In favor for the latter
assumption are reports that a blockade of the ATP site by
FITC modification of K501 inactivates the overall Na
+
/
K
+
ATP hydrolysis, but not the K
+
activated phosphatase
[13,17]. Because the ATP site is blocked by FITC (Fig. 3),
an ad ditional phosphatase site outside the A TP site might
exist in the isolated loop protein.
In fact, in silico docking experiments revealed that the
H
4
-H
5
loop may contain separate binding sites for ATP and
pNPP (Figs 5 and 6). Within the isolated H
4
-H
5
loop, the
ATP site of the N-domain retains no ATPase activity
[34,36]. Most likely this is due to the fact that the Mg
2+
binding site necessary for t he phosphorylation o f D369
[10–12,33] from both substrates resides on the P-domain’s
C-terminal part [61] and that N- and P-domains do not

bend together when the H
4
-H
5
loop is disconnected from
the transmembrane helices. Removal of the C-terminal
sequence of the P-domain is without effect on the
p-nitrophenylphosphatase activity (Table 1). Although
pNPP may interact with the ad enosine subsite of the ATP
site (Figs 5A,C and 6A,C), modification of this site by FITC
(Fig. 3) had only a minor effect on the pNPP activity
(Table 2). Additionally, and contrary to the expectations
from the literature reporting an inhibition of K
+
activated
phosphatase by ATP in t he full en zyme [31,60], even
millimolar concentrations of ATP did not affect the
phosphatase (data not shown). All these findings support
the conclusion that in the i solated H
4
-H
5
loop, pNPP is not
hydrolyzed via the ATP site although it may be able to bind
there (Figs 5A and 6A).
Structural models describing the three-dimensional fold-
ing of the a subunit f orming the H
4
-H
5

loop greatly
facilitate the interpretatio n of experimental findings. Unfor-
tunately, the str ucture of the H
4
-H
5
loop seems t o vary
depending on the p resence of the phosphorylation domain.
A model based on the E1 crystal structure of Ca
2+
ATPase
[19] and respecting t he N- and C-terminal parts of the
P-domain [20], shows F548 as part of the ATP site
(Fig. 3A), while in a model based on the crystal structure
of the N-domain of Na
+
/K
+
ATPase and not including the
P-domain [21] (Fig. 3B), F548 is buried under the surface of
the ATP site. The influence on F548 on ATP and TNP-ATP
binding has been i nvestigated [44,56,62]. The effect of
mutation of this specific amino acid on the nucleotide
binding property of the isolated H
4
-H
5
loop is rather drastic
[41]. It r emains, however, unclear whether F548 directly
interacts with ligands or whether its importance lies in the

formation of the structural backbone of the ATP binding
pocket. The adjacent amino acid residue C549, was shown
to be labeled by erythrosin isothiocyanate in the membrane-
embedded Na
+
/K
+
ATPase after blocking of the E
1
ATP
binding site with FITC [16]. Modification of this site with
the sulfhydryl- reactive 8-thiocyano-ATP forming a mixed
disulfide bridge may inactivate Na
+
/K
+
ATPase [63]. In
the H
4
-H
5
loop model of Na
+
/K
+
ATPase [20] obtained
analogously to E1-Ca
2+
ATPase [19], F548 is part of the
ATP binding site and C549 is accessible by induced fit [7];

whereas in the crystal structure derived exclusively from the
N-domain [ 21], both amino acids a re hidden under the
surface as part of the structural backbone. Hence, the model
respecting the P-domain-forming peptide extensions of the
N-domain [20] seems to fit better to the experimental data
(see below).
The p referred binding site for pNPP was found at a
specific surface at the rear side of the ATP binding pocket
on the N-domain (pNPP site; Fig. 5B,D). Docking experi-
ments revealed that pNPP binds to this site with a slightly
higher interaction energy than to the nucleotide binding site.
Unexpectedly, however, in silico do cking o f p NPP t o a
model based on the crystal structure of the N-domain of
Na
+
/K
+
ATPase [21,23] (Figs 5D and 6D) gave a different
location of the bin ding site than a model including the
P-domain [20] (Figs 1A, 5B a nd 6B). Docking of the
substrate to the latter model [20] revealed that pNPP binds
Table 3. Comparison of the primary structures of isoforms of Na
+
/K
+
ATPase and H
+
/K
+
ATPase in the range of the binding site for pNPP. Bold,

amino acids interacting with pNPP (see Fig. 6B); italic, amino acids not conserved in the compared sequences; dashes, amino acids not present in
the sequence.
Ó FEBS 2004 Mapping of ATP and pNPP binding sites of the Na pump (Eur. J. Biochem. 271) 3933
to a site i n close vicinity to N398, a residue that has been
affinity-labeled by 4-azido-2-nitrophenylphosphate in the
membrane-embedded N a
+
/K
+
ATPase [51]. The model
showing exclusively the isolated N-domain [21,23] (Figs 5D
and 6D), refused to dock pNPP close to N398. The new
position found for pNPP binding is 16 A
˚
away from N398
and shows a lower interaction energy than to the site in the
other model. Apparently, the model respecting N- and
C-terminal peptide extensions of the N-domain [20] des-
cribes more adequately the experimental findings (Figs 5B
and 6B): mutation of N398 to aspartate and truncation of
the P-domain’s N-terminal part, caused a drop of the
phosphatase activity (Fig. 4). The latter finding points to a
stabilizing effect o f the N-terminal sequence in forming the
pNPP site in the neighborhood of N398. The hydrolysis of
pNPP does not need the phosphorylation site D369 (Fig. 4).
The finding of a phosphatase site around N398 outside of
the ATP binding site, which is involved in the overall
Na
+
/K

+
ATPase [51], is puzzling. A comparison of the
amino acid sequences between N377 and L414 for various a
subunit isoforms of Na
+
/K
+
ATPase and H
+
/K
+
ATPase
revealed t hat N398 is conserved i n the a
1
subunit in
vertebrates only but not in insects and invertebrates
(Table 3). N398 probably interacts with the nitro group of
pNPP within the pNPP site. This amino acid residue,
however, is not present in the a
2
, a
3
and a
4
isoforms of
Na
+
/K
+
ATPase and does not seem to be necessary for the

phosphatase activity in H
+
/K
+
ATPase [31] (Table 3).
Tyrosine phosphorylation can generally be achieved from
pNPP [49], but phosphotyrosine is not hydrolyzed by the
isolated H
4
-H
5
loop. Because Mg
2+
is needed for pNPP
hydrolyis by the isolated H
4
-H
5
loop, the pNPP site close to
N398 seems to recognize this divalent cation. It is an open
question w hether it participates in the recently reported
MgATP dependent interaction of isolated H
4
-H
5
loops [55].
In conclusion, our data show that the isolated H
4
-H
5

loop of Na
+
/K
+
ATPase contains a single ATP site only
and an additional pNPP site around N398 that hydrolyzes
pNPP in a Mg
2+
dependent manner. The properties of
this additional site for pNPP served as a tool to compare
two different structure models of the isolated H
4
-H
5
loop
of Na
+
/K
+
ATPase. The model derived from the crystal
structures of E1-Ca
2+
ATPase [19] and respecting peptide
extensions of the N-domain forming the phosphorylation
domain [20], describes in a better way the effects of
truncation and single amino acid mutations on the
pNPPase (Figs 4 and 6B, Table 1) than a model [23]
referring to the crystal structure of N-domain alone [21]
(Fig. 5 D).
Acknowledgements

We thank Dr K.O. Ha
˚
kansson for providing the atomic coordinates of
the Na
+
/K
+
ATPase N-domain crystal structure.
This work was supported by the German and C zech Governments
through TSR-088-97, and CZE 00/33, by the Ministry of Education,
Youth and Sports of the Czech Republic (LN 00A141), by grants no.
204/01/0254, 204/01/1001, 206/03/D082, 309/02/1479 and MSMT
113100003 and the research projects no. AVOZ11922 of the Grant
Agency of the Czech Republic and the Deutsche Forschungsge me-
inschaft, Bonn Scho 139/21-2+3. The contributions of Drs T. Obs
ˇ
il
and M. Kubala to this work are gratefully acknowledged.
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