From the Department of Biochemistry and Molecular
Biology, Oregon Health and Science University, Portland, Oregon
97239 and the ¶ Shemyakin-Ovchinnikov Institute of
Bioorganic Chemistry, Russian Academy of Sciences,
117997 Moscow, Russia
Received for publication, January 2, 2003
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ABSTRACT |
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The copper-transporting ATPase ATP7B is essential
for normal distribution of copper in human cells. Mutations in
ATP7B lead to Wilson's disease, a severe disorder with
neurological and hepatic manifestations. One of the most common disease
mutations, a H1069Q substitution, causes intracellular
mislocalization of ATP7B (the Wilson's disease protein, WNDP).
His-1069 is located in the nucleotide-binding domain of WNDP and is
conserved in all copper-transporting ATPases from bacteria to mammals;
however, the specific role of this His in the structure and function of
WNDP remains unclear. We demonstrate that substitution of His-1069 for
Gln, Ala, or Cys does not significantly alter the folding of the WNDP
nucleotide-binding domain or the proteolytic resistance of the
full-length WNDP. In contrast, the function of WNDP is markedly
affected by the mutations. The ability to form an acylphosphate
intermediate in the presence of ATP is entirely lost in all three
mutants, suggesting that His-1069 is important for
ATP-dependent phosphorylation. Other steps of the WNDP
enzymatic cycle are less dependent on His-1069. The H1069C mutant shows
normal phosphorylation in the presence of inorganic phosphate; it binds
an ATP analogue, Copper is a cofactor of important metabolic enzymes that are
involved in a variety of physiological processes such as radical detoxification, oxidative phosphorylation, and iron metabolism. The key
role in copper distribution in human cells belongs to the
copper-transporting ATPases ATP7A and ATP7B (Menkes disease and
Wilson's disease proteins, respectively). These large polytopic membrane proteins utilize the energy of ATP hydrolysis to transport copper from the cytosol into the lumen of the secretory compartment where copper can be incorporated into various
copper-dependent enzymes (1). When intracellular copper
exceeds a certain level, the copper-transporting ATPases traffic to the
plasma membrane where they export excess copper out of the cell (2,
3). Mutations in ATP7A and ATP7B
result in disruption of copper transport from the cytosol and severe
pathologies in humans, i.e. Menkes disease and Wilson's
disease, respectively.
Menkes disease and Wilson's disease proteins
(MNKP1 and WNDP) belong to a
family of the P-type ATPases. The primary structure of MNKP and WNDP
include several signature sequences (Fig. 1A) that are
essential for enzymatic activity of all members of this family. The
ATP-binding domain of WNDP and MNKP also contains a sequence SEHPL,
which is conserved in all ATPases transporting transition metals (the
P1-type ATPases or CPx-ATPases) but not in other P-type
ATPases (Fig. 1). In the SEHPL sequence, His is an invariant residue
(Fig. 1B, alignment), suggesting that this amino acid is
essential for function or structure of the P1-type ATPases.
Significantly, substitution of His-1069 in WNDP for Gln is the
most frequent cause of Wilson's disease in northern European populations (4), an observation that underscores the importance of
His-1069.
Recent studies have shown that mutations of His-1069 to Gln or Ala
result in decreased intracellular stability of WNDP and retention of
the mutants in the endoplasmic reticulum (5, 6); the latter effect can
be overcome by lowering the temperature at which the cells are grown
(5). These results suggested a role for His-1069 in folding and
stability of WNDP. In contrast, replacement of the equivalent His
residue in MNKP, which is 60% homologous to WNDP, does not alter the
protein steady-state levels and does not disrupt normal targeting of
MNKP to the trans-Golgi network (7), indicating that in MNKP
the structural consequences of His replacement are minor. Thus, it
remains unclear whether His-1069 plays an important role in the
structural organization of WNDP.
The effect of the H1069Q substitution on the transport function of WNDP
was analyzed by the ability of the mutant protein do the following: (i)
complement the growth defects of a To elucidate the functional role of His-1069, we substituted His-1069
for various amino acid residues and characterized the effects of these
mutations on WNDP folding and enzymatic activity. Our experiments
indicate that His-1069 is important for a specific step in the WNDP
catalytic cycle, phosphorylation from ATP, and is not critical for
other steps of the enzymatic reaction or for overall protein folding.
His-1069 appears to control, directly or indirectly, positioning of ATP
in the active site of WNDP.
Generation of the His-1069 Mutants of the Full-length WNDP and
Expression in SF9 Cells--
The H1069Q, H1069A, and H1069C mutants of
WNDP were generated using polymerase chain reaction. The following
forward and reverse primer pairs were used to introduce His
The generated plasmids were then utilized to produce recombinant
baculoviruses; the wild-type (wt) and mutant WNDPs were then expressed
using virus-mediated infection of SF9 cells as described previously
(10). Expression of the WNDP mutants was verified by SDS-PAGE and
Western blot analysis with anti-WNDP antibodies; the amount of
expressed protein was determined by densitometry of the
Coomassie-stained bands. The level of expression for mutant proteins
was, on average, ~50-70% of wt WNDP. The membrane preparations of
insect cells expressing either wt or mutant WNDP were isolated according to Ref. 10; the membrane protein was stored at WNDP Phosphorylation from 32Pi Inorganic
Phosphate--
50 µg of membrane protein preparation was resuspended
in 200 µl of a buffer containing 50 mM MES-Tris (pH 7.0),
10 mM MgCl2, 20% Me2SO
(Pi-buffer), and then 0.66 µl of 100 µCi of
32Pi (specific activity, 6000 Ci/mmol) was
added to the mixture. Following a 10 min incubation at room
temperature, 50 µl of 1 mM NaH2PO4 in 50%
(w/v) trichloroacetic acid was added to stop the reaction, and
precipitated proteins were collected by centrifugation at 20,000 × g for 10 min and then rinsed with 1 ml of cold
H2O. The protein was resuspended in the sample buffer (5 mM Tris-PO4, pH 5.8, 6.7 M urea,
0.4 M dithiothreitol, 5% SDS), and incorporation of
32P into WNDP was analyzed on acidic SDS-PAGE as described
(10).
To determine the effect of ATP and the non-hydrolyzable ATP analogue
To determine the effect of copper on phosphorylation from
Pi, wt and mutant WNDPs were incubated at room temperature
in Pi buffer containing 100 µM
Tris-(2-carboxyethyl)phosphine (TCEP) and 100 µM
ascorbate in the absence or presence of 250 µM copper chelator bathocuproine disulfonate (BCS) and then phosphorylated by
Pi as described above. To reverse the effect of the
chelator, 135 µM CuCl2 was added to the WNDP
sample preincubated with 250 µM BCS. BCS binds copper
with a stoichiometry of 2:1; thus this procedure generates 10 µM free copper in a solution. Following 10 min of
incubation at room temperature, the Pi phosphorylation reaction was then carried out as described above.
Generation of the ATP-binding Domain Mutants and Expression of
Recombinant Proteins in Escherichia coli--
The expression plasmid
pET28b-ATP-BD encoding the ATP-binding domain of WNDP (ATP-BD, amino
acid residues Lys-1010 to Ly-1325) was described previously (11). To
introduce various mutations of His-1069 into the recombinant ATP-BD,
the Bsu36I-PstI fragments of the mutant full-length WNDP
cDNA generated as described above were excised from the
pFastBacDual-WNDP plasmid and cloned into the pET28b-ATP-BD plasmid
digested with the Bsu36I and PstI restriction endonucleases. Expression in E. coli and purification of
mutant ATP-BDs were performed according to (11). Protein expression and
purity were verified by SDS-PAGE on 12% Laemmli gels.
Folding of the Wild-type and Mutant ATP-BDs--
The effect of
His-1069 substitutions on ATP-BD folding was analyzed using intrinsic
Trp fluorescence as described in (11) and by circular dichroism
spectroscopy (CD). The CD spectra in the region 180-260 nm were
recorded in 50 mM NaH2PO4, 250 mM NaF buffer at a protein concentration of 0.2 mg/ml using
an AVIV CD model 215 spectrometer. The protein concentration was
verified by amino acid analysis of corresponding samples and further
used to calculate the Proteolityc Digestion of Full-length WNDP Expressed in Insect
Cells--
10 µg of membrane preparations from insect cells
expressing the wt WNDP or His mutants was resuspended in 20 µl of 25 mM imidazole, pH 7.4, and 250 mM sucrose
buffer. Tosylphenylalanyl chloromethyl ketone (TPCK)-treated trypsin
(Sigma) was added up to 0.83 µg/ml, and the reaction mixture was
incubated at room temperature for 30 min. The proteolytic reaction was
terminated by addition of the protease inhibitor
4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) to 2 µM.
The reaction mixture was then diluted to 100 µl with SDS-PAGE loading
buffer (100 mM Tris, pH 6.8, 3.3% SDS, 2.6 M
urea, 5% Molecular Modeling--
The high resolution structure of the
ATP-binding domain of SERCA1 Ca2+-ATPase (residues Ala-320
to Lys-758) was utilized as a template to generate a molecular model
for the ATP-binding domain of WNDP (residues Met-996 to Arg-1322). The
ATP-binding domain of WNDP is composed of segments Val-997 to Ala-1065,
Asp-1185 to Ile-1236, and Phe-1240 to Ile1311, which share significant
homology with Ca2+-ATPase. Segment Ser-1066 to Ile-1184 of
ATP-BD is located between these homologous regions and has little
similarity to the corresponding region of Ca2+-ATPase. The
secondary structure predictions for WNDP and subsequent search in the
data base of high-resolution structures using threading algorithms
revealed that the predicted fold of the ATP-binding domain of WNDP
matches unequivocally to the fold of the Ca2+-ATPase
ATP-binding domain in both conserved and non-conserved regions. The
alignment of sequences and secondary structures generated by the
GenTHREADER program (13) served as a basis for the ATP-BD model. The
model was built using Modeler software (14) and included thorough loop
optimization. 25 models with different loop conformations were
generated. The model with the highest quality score, as defined by the
Profiles_3D program (15), was selected as the best one. The above
procedure was employed to build ATP-BD models using three structural
templates, i.e. the high-resolution structures of
Ca2+-ATPase in the E1 and E2 states (Protein Data Bank
codes 1EUL (16) and 1IWO (17), respectively) as well as electron
microscopy structure in the E2 state (Protein Data Bank code 1KJU
(18)).
The Effect of the His-1069 Substitution on Folding of the
ATP-binding Domain--
As shown in Fig.
1, His-1069 is located in the ATP-binding
domain of WNDP (ATP-BD) and could be essential for correct folding of
this important functional domain. To test this hypothesis, we expressed
and purified the wt ATP-BD and ATP-BDs in which the His residue was
replaced with one of the following amino acid residues, Gln, Ala, and
Cys. Analysis of protein folding using CD spectroscopy revealed that
overall secondary structure of all mutants
is similar to the wt ATP-BD structure
(Fig. 2A, and Table I),
indicating that the mutations do not cause marked misfolding of this
domain.
This conclusion was further verified using intrinsic Trp fluorescence.
ATP-BD contains a single Trp residue, which has a buried position in
the wt protein (11). Unfolding of ATP-BD results in a
decrease of Trp fluorescence intensity and a red shift of the fluorescence maximum (11). Fig. 2B illustrates that even the bulkiest H1069Q substitution does not have a significant negative effect on intrinsic Trp fluorescence of ATP-BD, confirming that His-1069 is not critical for ATP-BD folding. This conclusion was at
odds with recent cellular studies wherein decreased stability and
mislocalization of the H1069Q mutant pointed to somewhat abnormal folding of the mutated WNDP (5). Consequently, we hypothesized that
mutations of His may alter domain-domain interactions within the
full-length WNDP rather than folding of the individual domains. The
disruption of this interaction may expose some protein regions to
cellular proteases and thus decrease protein stability.
To determine the effect of His substitution on folding of the
full-length WNDP, the Gln, Cys, and Ala substitutions of
His-1069 were introduced into the full-length WNDP, and the
mutants were expressed in insect cells using baculovirus-mediated
infection of SF9 cells. As shown in Fig.
3A, the steady state protein
levels were similar for all three mutants and not markedly different from the protein levels of the wt WNDP (~50-70% of the wt). More importantly, we did not observe significant fragmentation of any of the
WNDP mutants, suggesting that the expressed proteins are fairly stable
(Fig. 3B). To further evaluate stability of the expressed
proteins in vitro, the membrane fractions containing the wt
and mutant WNDPs were prepared and subjected to limited tryptic
digestion, and the proteolytic resistance of the mutants and wt WNDP
was compared. Fig. 3B illustrates that the patterns of
proteolytic fragments for the wt and mutant WNDPs are very similar. In
some experiments we observed a slight difference in relative abundance
of the produced fragments between the mutant and wt WNDPs, suggesting
that the replacement of His made the contacts between different regions
of the protein somewhat more flexible or exposed. There was also a
subtle difference in the proteolytic resistance of the three mutants.
Consistently, the H1069A mutant was less resistant than the H1069Q and
H1069C mutants, whereas the latter mutant appeared to be the most
resistant. Overall however, it is clear that the folding of WNDP is not
grossly affected by the His-1069 mutations.
The Catalytic Phosphorylation of WNDP by ATP Is Disrupted by
Mutation of His-1069--
As we demonstrated previously, WNDP
functions as a copper-dependent P-type ATPase,
i.e. during its catalytic cycle WNDP transfers the
As shown in Fig. 4A, all three
mutants demonstrated markedly reduced phosphorylation activity, which
did not exceed that of the inactive D1027A mutant. Catalytic
phosphorylation of the mutants could not be detected even when
sensitivity of the assay was increased by raising the specific
radioactivity of [ His-1069 Is Not Essential for Phosphorylation of WNDP by Inorganic
Phosphate--
The characteristic feature of the P-type ATPases is
their ability to form a phosphorylated catalytic intermediate when
incubated with inorganic 32P phosphate in the presence of
magnesium. This reaction reflects the reversibility of the
dephosphorylation step in the enzyme catalytic cycle. The
conformational state of the P-type ATPases, which favors
phosphorylation from Pi (the E2-state), is
different from the one favoring phosphorylation from ATP (the
E1-state). Consequently, we used phosphorylation from
Pi to test whether the His-1069 mutations eliminated
phosphorylation in general or specifically affected the
ATP-dependent step of the catalytic cycle. This experiment
produced an interesting result (Fig. 4, B-D).
The H1069Q and H1069A mutants demonstrated markedly reduced level of
phosphorylation from Pi (~5-10%). In contrast, the
H1069C mutant was phosphorylated at levels close to the wt
phosphorylation (80-90%). These results indicate that His-1069 is not
essential for phosphorylation of WNDP by PI; however, the
1069 position plays an important role in the environment of the
catalytic site.
In the P-type ATPases, phosphorylation from Pi takes place
when the exported ion is released from the transport site, allowing the
protein to adopt the E2 conformation. Accordingly, addition of the
transported ion to the reaction prevents formation of the E2 state and
decreases the Pi-mediated phosphorylation. This property of the P-type
ATPases was used to test whether the His mutations have an effect on
the ability of WNDP to bind copper at the site(s) important for
catalytic phosphorylation. As shown in Fig.
5, addition of the copper chelator BCS to
the wt WNDP stimulates phosphorylation of WNDP from Pi,
whereas the addition of copper inhibits the phosphorylation reaction in
agreement with the predicted properties of WNDP as a P-type ATPase.
Importantly, the His-1069 mutants behaved in these reactions similarly
to the wt WNDP, confirming that the His substitutions do not compromise
the ability of mutants to bind copper.
Nucleotide Binding Properties and Conformational Transitions of the
H1069C Mutant--
Further studies were carried out with the H1069C
mutant, which showed close to normal phosphorylation from
Pi and normal response to copper additions but could not be
phosphorylated from ATP. One explanation for this phenotype is that
binding of ATP is disrupted by the mutation. Alternatively, the H1069C
mutant can bind ATP but fails to undergo conformational transitions in
response to nucleotide binding. For WNDP, direct measurements of ATP
binding are still technically difficult. Consequently, to examine the above possibilities, we carried out limited proteolysis of WNDP in the
presence of increasing concentrations of a non-hydrolyzable ATP
analogue, AMP-PNP. As shown in Fig.
6A, AMP-PNP alters the proteolytic pattern for both the wt WNDP and the H1069C mutant, making
several sites less accessible to proteolytic digestion. Moreover, the
same protein regions of the wt WNDP and the mutant became resistant to
proteolysis, suggesting that the major structural changes upon binding
of AMP-PNP are similar for both proteins. Thus, the lack of
ATP-dependent phosphorylation in the case of the H1069C
mutant is unlikely due to lack of ATP binding.
The proteolysis experiments provide only a rough estimate of the
ability of WNDP and the His mutant to bind the nucleotide. It is quite
possible that mutations of His-1069 alter the positioning of ATP within
the catalytic site rather than disrupt the nucleotide binding. If this
is the case, one may expect to see quantitative differences in the
nucleotide-binding characteristics of wt WNDP and the H1069C mutant. To
characterize ATP binding more quantitatively, we incubated WNDP and the
H1069C mutant with increasing concentrations of the non-hydrolyzable
ATP analogue (AMP-PNP) and then measured phosphorylation from
Pi. Binding of the nucleotide was expected to stabilize the
enzyme in the E1-like state and therefore decrease efficiency of the
Pi-mediated phosphorylation. As shown in Fig. 6B, the addition of AMP-PNP inhibits incorporation of
32Pi for both the wt WNDP and H1069C mutant;
however, the efficiency of inhibition is different. The apparent
Ki values for the wt WNDP and the H1069C mutant are
equal to 116 ± 19 µM and 533 ± 166 µM, respectively, suggesting that the H1069C mutant binds
AMP-PNP less effectively.
Finally, we compared the effects of ATP and AMP-PNP on phosphorylation
by 32Pi. ATP is expected to allow enzyme
turnover, producing an additional decrease in 32P
incorporation compared with AMP-PNP. As shown in Fig. 6C,
the addition of ATP to the wt WNDP indeed leads to a larger inhibition of the 32Pi-incorporation than a
non-hydrolyzable analogue. In the case of the H1069C mutant, ATP and
AMP-PNP have a similar effect on the Pi-mediated
phosphorylation, suggesting that ATP binds to the mutant but is not hydrolyzed.
Spatial Location of His-1069 in the ATP-binding Domain--
To
better visualize the position of His-1069 in the ATP-binding domain, we
generated a molecular model of the ATP-binding domain using the
recently published high resolution structure of Ca2+-ATPase
in the E2-conformation (17). In this conformation, the nucleotide-binding region (the N-domain) and the phosphorylation region
containing the DKTG and GDGXXD motifs (the P-domain) come fairly close together in contrast to the Ca2+-bound form in
which these two subdomains are located far apart (16). Although the
E2-state structure cannot be used to predict the exact position of ATP
prior to hydrolysis, the model based on this structure provides a
useful guide to relative distances and mutual positions of the residues
in the ATP-binding domain. The generated model is shown in Fig.
7. It revealed that the spatial location
of the SEH1069PL sequence is equivalent to that of the
438GEAT441 segment of Ca2+-ATPase.
The 438GEAT441 segment is a part of the
nucleotide-binding region of Ca2+-ATPase and was shown to
be in direct proximity to bound ATP (20). Furthermore, even
in the E2 state His-1069 is only 18Å away from the Asp residues of the
GDGVND motif that are known to be close to the WNDP has a central role in human copper homeostasis; however, the
molecular mechanism of the WNDP-mediated copper transport remains
poorly understood. Until recently, the functional characterization of
this protein has been hindered by the lack of an expression system
suitable for biochemical investigations. Recent development of
functional expression of WNDP in insect cells (10) opened the door to
analysis of the molecular mechanism of WNDP and supplied necessary
tools for understanding how disease-causing mutations affect folding,
function, and regulation of WNDP.
To provide detailed characterization of the Wilson's disease
mutations, in this work we expanded our earlier analysis of the enzymatic properties of WNDP. We demonstrated that WNDP can be phosphorylated in the presence of inorganic phosphate and magnesium and
that this reaction is facilitated upon removal of copper by BCS and is
inhibited by copper addition. We also found that binding of nucleotides
induces conformational changes in WNDP. These properties confirmed that
the overall catalytic cycle of WNDP resembles those of a typical P-type
ATPase. The developed tools were then utilized to characterize the role
of His-1069 in the structure and function of WNDP.
His-1069 is particularly interesting because it is a site of the most
frequent mutation in Wilson's disease patients and an invariant
residue in the P1-type ATPases. His-1069 is located in the
ATP-binding domain of WNDP, a protein region that is most conserved in
all P-type ATPases. The ATP-binding domains of several P-type ATPases
have been extensively characterized, and chemical modification,
mutagenesis, and NMR studies have yielded a detailed map of residues
important for nucleotide binding in Ca2+-ATPase (20).
However, this information has only limited value for understanding the
role of His-1069, because this residue lies in the region of the
ATP-binding domain (the so called N-subdomain), the sequence of which
is unique for the P1-type ATPases. No equivalent histidine
is present in the ATP-binding domain of Ca2+-ATPase or
other well characterized P2-type ATPases. Conversely, several residues known to play an important role in the binding of ATP
in Ca2+-ATPase and other P2-ATPases are absent
in WNDP and its homologues. This raises an interesting possibility
that, despite overall similarity of the catalytic cycles, the
P1-type ATPases and P2-type ATPases have
distinct subsets of amino acid residues involved in ATP-coordination and possibly catalysis. The results of our experiments support this hypothesis.
The first important result of this work is the demonstration that
folding of either the isolated ATP-binding domain or the full-length
WNDP is not significantly altered by mutations of His-1069. These
results argue against a significant role for this residue in the
overall structure of WNDP and seem to be at odds with recent studies
showing decreased intracellular stability of the His-1069 mutant
(5). Two considerations may help to reconcile this apparent
discrepancy. First, although we observed only a slight increase in
proteolytic sensitivity of the His-1069 mutants compared with the wt
WNDP, it seems likely that in a cell even small changes in the folding
are sufficient to decrease protein stability over time. Second, in the
cellular studies the negative effect of the H1069Q mutation on
intracellular localization of WNDP was eliminated at room temperature
(5), suggesting that, at temperatures lower than 37 °C, the folding
of WNDP was sufficient to pass quality control in the endoplasmic
reticulum. For our studies, we produce WNDP in the insect cells, which
grow at 27 °C. Therefore, it seems likely that folding of the WNDP
mutants in insect cells is better than in mammalian cells. This
unexpected benefit of the protein expression in SF9 cells helped us to
marginalize the effect of the His mutations on the WNDP structure and
dissect the functional role of His-1069.
Functional analysis of the His-1069 mutants revealed that His is not
essential for the dephosphorylation step of the catalytic cycle as
evidenced by the normal level of Pi-mediated
phosphorylation of the H1069C mutant. Mutations of His-1069 do not
disrupt copper binding to the site(s) that control catalytic
phosphorylation. Similarly, ATP binding by the mutant, although
diminished, is not critically affected. This last result is consistent
with the lack of significant structural changes in ATP-BD, where ATP
coordination is likely to be provided by several amino acid residues.
In striking contrast, phosphorylation from ATP is markedly decreased in
the mutants, suggesting that His-1069 plays an important role during this step of the catalytic cycle.
To better understand the role of His-1069 in ATP-dependent
phosphorylation, it is useful to compare the results of our work with
recent studies on bacterial homologues of WNDP. The histidine residues
equivalent to His-1069 have been mutated in two bacterial P1-type ATPases, a copper-transporting ATPase CopB (22) and a zinc and cadmium-transporting ATPase, ZntA (23). In CopB, the His The resolution of our methods is currently insufficient to distinguish
between direct involvement of His-1069 in coordination of ATP and a
more indirect role in which His-1069 interacts with other residues in
WNDP and helps to organize the catalytic site by forming specific
protein-protein contacts. It seems likely that, prior to transfer of
the In summary, we characterized the role of the invariant His-1069 in the
structure and function of WNDP. We conclude that His-1069 is important
for catalytic activity of WNDP and is likely to be involved in
positioning of ATP prior to transfer of the ,
-imidoadenosine 5'-triphosphate (AMP-PNP), and
copper and undergoes nucleotide-dependent conformational transitions similar to those of the wild-type WNDP. Although binding of
AMP-PNP is not disrupted by the mutation, the apparent affinity for the
nucleotide is decreased by 4-fold. We conclude that His-1069 is
responsible for proper orientation of ATP in the catalytic site of WNDP
prior to ATP hydrolysis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ccc2 yeast strain
lacking the WNDP homologue Ccc2 (8, 9); and (ii) decrease the toxic
effects of copper on growth of fibroblasts in cell culture (5). These
experiments demonstrated that mutations of His-1069 impaired the
transport function of WNDP. However, the specific role of this
invariant histidine in copper transport by WNDP has not been explained.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Gln,
His
Ala, and His
Cys substitutions, respectively:
5'-AGCAGTGAACAACCCTTGGGCGTGG and 5'-ACGCCCAAGGGTTGTTCACTGCTGG;
5'-AGCAGTGAAGCTCCCTTGGGCGTGG and 5'-ACGCCCAAGGGAGCTTCACTGCTGG; and
5'-AGCAGTGAATGCCCCTTGGGCGTGG and 5'-ACGCCCAAGGGGCATTCACTGCTGG. The
oligonucleotides: 5'-GGTATGGATTGTAATCGG-3' and
5'-CGTCGACGCCTGCCTGAA-3' were used as a forward and reverse flanking
primers. Following PCR, the fragments containing mutations were
digested with the ClaI and SalI restriction
endonucleases and the ClaI-SalI fragments were
exchanged with the corresponding fragment of the wild-type,
full-length WNDP cDNA cloned into the pFastBacDual-WNDP plasmid.
Generation of the pFastBacDual-WNDP plasmid has been described
previously (10). The presence of the mutations in the final construct
and the absence of unwanted mutations were confirmed by automated DNA sequencing.
80 °C in
a buffer containing 25 mM imidazole, pH 7.4, and 250 mM sucrose until further use. Analysis of WNDP
phosphorylation in the presence of [
-32P]ATP was
carried out as described in Ref. 10.
,
-imidoadenosine 5'-triphosphate (AMP-PNP) on phosphorylation of
WNDP by 32Pi, 50 µg of membrane preparation
containing either wt or mutant WNDP was pre-incubated in
Pi-buffer with increasing concentrations of the nucleotide
(10 µM-2 mM) for 10 min at room temperature, and then Pi-mediated phosphorylation was carried out as
described above.
values. The secondary structure contents
in WNDP and its mutants were estimated using the set of 43 reference protein spectra and the following algorithms of CD spectra
deconvolution: CONTIN, SELCON3, CDSSTR, and CDNN (12).
-mercaptoethanol), and 10 µl of the mixture was loaded
onto a 7.5% Laemmli gel. Following separation and Western blotting,
the WNDP proteolytic pattern was analyzed by immunostaining using
anti-ATP-BD antibody as described previously (10). To determine the
effect of the nucleotide binding on proteolytic sensitivity of WNDP and
the H1069C mutant, 10 µg of respective membrane preparations were
incubated in 20 µl of 25 mM imidazole, pH 7.4, and 250 mM sucrose buffer with an increasing concentration of
AMP-PNP (0-50 µM) for 10 min at room temperature.
Trypsin was then added, and the proteolytic digestion and analysis of
the fragments were performed as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of the transmembrane
organization of WNDP. A, the letters TGEA,
TGDN, DKTG, and GDGVND indicate
sequence motifs conserved in all P-type ATPases. The bold
letter D in the DKTG motif marks the position of
Asp-1027, an acceptor of Pi during catalysis. The SEHPL
sequence is conserved in all P1-type ATPases and contains
the invariant His-1069, marked by larger font. The CXXC
motifs indicate copper-binding sites in the cytosolic copper-binding
domain. B, the alignment of the ATP-binding domain segments
of several P1-type ATPase. The alignment was generated
using ClustalW (www.ebi.ac.uk/clustalw/). The protein data base
accession numbers are given in parentheses for the following:
atp7a_human, MNKP (Q04656); atp7b_human, WNDP (P35670); atu2_yeast,
yeast copper-transporting ATPase CCC2 (P38995); atzn_ecoli, lead-,
cadmium-, and zinc-transporting ATPase (P37617); cada_stauu,
cadmium-transporting ATPase (P20021); copa_enthr, copper-importing
ATPase A from Enterococcus hirae (P32113); and
copb_enthr, copper-exporting ATPase B from E. hirae (P05425). The invariant His is in bold; the
SEHPL-like sequences are underlined.
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Fig. 2.
Folding properties of the wt and mutant
ATP-binding domains. A, Circular dichroism spectra for
wt ATP-BD (WT) and the H1069Q (HQ), H1069A
(HA), and H1069C (HC) mutants were recorded at a
protein concentration of 0.2 mg/ml in buffer containing 50 mM NaH2PO4 and 250 mM
NaF, pH 7.0. B, the fluorescence emission spectra of
wt ATP-BD (WT), the H1069Q mutant (HQ), and
L-tryptophan (Trp) at equimolar
concentrations.
Secondary structure composition of ATP-BD (WT) and the ATP-BD
mutants with His-1069 substituted for Ala (HA), Cys (HC), or Gln
(HQ)
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Fig. 3.
Expression of the His-1069 mutants using
baculovirus-mediated infection of insect cells and analysis of their
proteolytic stability. A, Coomassie staining of the
membrane fractions isolated from SF9 cells infected with empty virus
(Mock), virus expressing wt WNDP (WT), or one of
the following mutants: H1069Q (HQ), H1069C (HC),
and H1069A (HA). 50 µg of total membrane protein is loaded
per lane. B, left panel, Western blot
analysis of 2 µg of membrane preparations containing either wt or
mutant WNDPs. Right panel, 10 µg of membrane preparation
containing indicated proteins were treated with trypsin as described
under "Experimental Procedures" and analyzed by Western
blotting
-phosphate from ATP to the invariant Asp-1027, generating a fairly
stable phosphorylated intermediate (10). Consequently, to evaluate the
effect of His-1069 mutations on WNDP function, we determined whether
the mutants were able to form a catalytic phospho-intermediate. Cell
membranes containing wt or mutant WNDP were incubated with
-[32P]ATP, and incorporation of 32P into
WNDP was monitored following separation of membrane proteins on an
acidic polyacrylamide gel (19). The catalytically inactive D1027A
mutant of WNDP was used in these experiments as a background control.
-32P]ATP 10-fold (data not shown).
His-1069 is located in the ATP-binding domain of the protein, and one
of the reasons for the observed effect could be a dramatic decrease in
the affinity of mutant WNDPs for ATP. To explore this possibility, the
ATP concentration in the phosphorylation assay was increased up to 1 mM, but no catalytic phosphorylation of any of the His-1069
mutants was detected. Similarly, changing other reaction conditions
(temperature, pH of the reaction in the range of 5.0-8.5, buffer
composition) did not lead to appearance of the acylphosphate
intermediate for any of the mutant WNDPs (data not shown). Altogether,
the results suggested that mutations of His-1069 had a strong negative
effect on the catalytic function of WNDP. Further experiments were
carried out to determine more precisely which step of the catalytic
cycle is most affected.
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Fig. 4.
Catalytic phosphorylation of the WNDP and
mutants using [ -32P]ATP and
32Pi. A and B, 50 µg of total membrane preparation of wild-type or mutant WNDP were
incubated with 1 µM of [
-32P]ATP (5 µCi; specific activity, 20 Ci/mmol) on ice at pH 7.0 for 4 min (for
phosphorylation from ATP) (panel A) or with 80 nM of 32Pi (100 µCi; specific
activity, 6000 Ci/mmol) at room temperature at pH 7.0 for 10 min (for
phosphorylation from Pi) (panel B). The
phosphorylated intermediate was detected by autoradiography following
separation of samples on an acidic gel. C, the gels were
then rehydrated and stained with Coomassie R250 (Protein).
Abbreviations for the mutants are the same as in the legend to Fig. 3.
The lane marked DA contains a sample with the
D1027A mutant. D, the incorporation of 32P from
inorganic phosphate normalized to the amount of WNDP determined by
densitometry.
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Fig. 5.
Effect of copper on phosphorylation of the
WNDP mutants in the presence of 32Pi inorganic
phosphate. Phosphorylation was measured under standard conditions
or following treatment of the membrane preparations with the copper
chelator BCS with or without subsequent addition of copper. The
comparison of the wt WNDP (WT) and two mutant WNDPs
(HC and HQ) are shown; each lane contains 50 µg
of membrane protein. The HA mutant was also tested and showed a similar
response.
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Fig. 6.
Effect of AMP-PNP or ATP on proteolytic
sensitivity (A) and on
32Pi-mediated phosphorylation (B
and C) of the wt WNDP and the H1069C
mutant. A, 10 µg of membrane protein was incubated
with increasing concentrations (0-50 µM) of AMP-PNP and
then proteolyzed with trypsin as described under "Experimental
Procedures." The fragments were separated on a 7.5% Laemmli gel and
visualized by immunostaining using antibody directed against the
ATP-binding domain of WNDP. B and C, membrane
preparations containing the wt WNDP (WT) and the H1069C
(HC) mutant were incubated with increasing concentrations of
AMP-PNP (panel B) or 200 µM ATP or
AMP-PNP (panel C) for 10 min at room temperature, and then
32Pi-mediated phosphorylation was carried out.
Following autoradiography, the intensity of the 32P-labeled
bands was quantified by densitometry.
-phosphates of the
ATP-magnesium complex during catalysis (21).
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Fig. 7.
A molecular model of the ATP-binding domain
of WNDP. The model is based on the high-resolution structure of
Ca2+-ATPase in the E2-state (Protein Data Bank accession
number 1IWO). The molecule is shown in a ribbon representation.
The SEHPL motif with the His-1069 side chain and the DKTG and GDGVND
motifs are shown in green. An ATP molecule (in
pink) at the top is shown to illustrate relative
distances between these three motifs. D1027 and
arrow indicate the position of Asp-1027, an acceptor of
-phosphate during ATP-dependent phosphorylation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Gln mutation drastically reduced the ATPase activity and decreased
phosphorylation from ATP by 80% (22). In a study of ZntA, introduction
of His
Gln mutation also decreased the ATPase activity but less
than in the case of CopA (37% of activity remained). The level of
phosphorylation from ATP and Pi in ZntA mutants was
reduced, and dephosphorylation was slowed down (23). In this earlier
work, the multiple consequences of the His substitution made it
difficult to assign a specific role to the histidine. Nevertheless,
these studies are very helpful. Together with our results, they
indicate that His strongly affects the environment in close proximity
to the phosphorylated Asp but is not essential for the chemistry of ATP
hydrolysis, because ATP-hydrolysis is not abolished in the bacterial
ATPase mutants. The mild decrease in the apparent affinity for AMP-PNP
and the drastic effect on phosphorylation from ATP observed in our
experiments further suggest that His-1069 plays a key role in orienting
ATP with respect to catalytic aspartate, thus allowing phosphorylation
to occur.
-phosphate, His-1069 directly orients ATP with respect to
catalytic Asp, whereas after the phosphorylation step the role of
His-1069 became less direct. This conclusion is consistent with the
predicted location of His-1069 in the ATP-binding domain (Fig. 7) and
is in agreement with our experimental data showing normal
PI phosphorylation by the H1069C mutant. The negative effect of the H1069A and H1069Q mutations on Pi
phosphorylation most likely stems from altered protein-protein contacts
within WNDP. It is interesting that H1069A substitution has a stronger negative effect on structure and function of WNDP than either H1069Q or
H1069C mutations, suggesting that the presence of a residue that can
form a hydrogen bond is tolerated better than the presence of the small
and neutral Ala.
-phosphate. Because His
in the SEHPL motif is an invariant residue in all P1-type
ATPases, it is plausible that this histidine has a similar role in all
these transporters. We also demonstrated that, at lower temperatures,
the folding of the common Wilson's disease mutant H1069Q is close to
normal but that this improvement of protein folding does not restore
catalytic activity of WNDP, an important conclusion for future
development of the corrective therapies for Wilson's disease.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Jens Peter Andersen and Dr. Michel Green for helpful discussion and for pointing out the similarities in the predicted spatial location of His-1069 and Glu-441 of SERCA1 Ca2+-ATPase. We also thank Kerry Maddox (Shriners Hospital for Children, Portland, OR) for performing amino acid analysis, Ms. Tina Purnat for help with preparation of figures, and Drs. D. Huster and J. H. Kaplan and Ms. Mee Min for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was funded by National Institutes of Health Grant DK55719 (to S. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of American Heart Association Postdoctoral Fellowship Grant 0120573Z.
Recipient of financial support from the Science Support
Foundation (Russia).
** To whom correspondence should be addressed. Tel.: 503-494-6953; Fax: 503-494-8393; E-mail: lutsenko@ohsu.edu.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M300034200
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ABBREVIATIONS |
---|
The abbreviations used are:
MNKP, Menkes disease
protein;
WNDP Wilson's disease protein, AMP-PNP,
,
-imidoadenosine 5'-triphosphate;
wt, wild-type;
MES, 4-morpholineethanesulfonic acid;
BCS, bathocuproine disulfonate;
ATP-BD, ATP-binding domain;
CD, circular dichroism.
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REFERENCES |
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