Introducing Wilson disease mutations into the zinc-transportingThe mutation P634L in the ÔhingeÕ motif GDGXNDXP perturbs the formation of the E2P state Juha Okkeri*, Eija Bencomo*, Marja
Trang 1Introducing Wilson disease mutations into the zinc-transporting
The mutation P634L in the ÔhingeÕ motif (GDGXNDXP) perturbs the formation
of the E2P state
Juha Okkeri*, Eija Bencomo*, Marja Pietila¨ and Tuomas Haltia
Institute of Biomedical Sciences/Biochemistry, University of Helsinki, Finland
ZntA, a bacterial zinc-transporting P-type ATPase, is
homologous to two human ATPases mutated in Menkes
and Wilson diseases To explore the roles of the bacterial
ATPase residues homologous to those involved in the
human diseases, we have introduced several point mutations
into ZntA The mutants P401L, D628A and P634L
corres-pond to the Wilson disease mutations P992L, D1267A and
P1273L, respectively The mutations D628A and P634L are
located in the C-terminal part of the phosphorylation
domain in the so-called hinge motif conserved in all P-type
ATPases P401L resides near the N-terminal portion of the
phosphorylation domain whereas the mutations H475Q and
P476L affect the heavy metal ATPase-specific HP motif in
the nucleotide binding domain All mutants show reduced
ATPase activity corresponding 0–37% of the wild-type
activity The mutants P401L, H475Q and P476L are poorly phosphorylated by both ATP and Pi Their dephosphory-lation rates are slow The D628A mutant is inactive and cannot be phosphorylated at all In contrast, the mutant P634L six residues apart in the same domain shows normal phosphorylation by ATP However, phosphorylation by Pi
is almost absent In the absence of added ADP the P634L mutant dephosphorylates much more slowly than the wild-type, whereas in the presence of ADP the dephosphorylation rate is faster than that of the wild-type We conclude that the mutation P634L affects the conversion between the states
E1P and E2P so that the mutant favors the E1or E1P state Keywords: P-type ATPase; Wilson disease mutation; heavy metal transport; hinge motif; ion translocation
P-Type ATPases form a large transporter protein family,
whose members share a few invariant or well-conserved
sequence patterns, although the overall degree of identity
among the sequences is rather low [1–3] A characteristic
feature is the presence of an aspartate in the sequence
DKTG; this residue is phosphorylated by ATP during the
catalytic cycle, hence the term P-type ATPases Sequence
motifs typical of the heavy metal transporting P-type
ATPases (P1-ATPases or CPx-ATPases) are presented in
Fig 1
The recently solved crystal structure of sarcoplasmic
Ca-ATPase [4] shows that the peripheral part of the enzyme
comprises three structural entities: the phosphorylation (P),
nucleotide binding (N) and actuator (A) domains Four
transmembrane (TM) helices contribute to the binding site
of two calcium ions inside the lipid bilayer The P domain, in
which the phosphorylated aspartate resides, is composed of
the N- and C-terminal parts of the longer cytoplasmic loop
(Fig 1), whereas the middle part of the loop makes up the N
domain The A domain with its conserved TGES motif
contains residues from the shorter cytoplasmic loop In the
crystal structure, both the N domain with the bound nucleotide and the TGES motif of the A domain are some
25 A˚ from the phosphorylated aspartate Likewise, the distance from the active aspartate to the bound substrate ions in the TM domain is long (about 50 A˚) Thus the structure does not directly reveal the molecular mechanism
of the ATP-powered ion translocation of the enzyme Because the bound ATP and the critical aspartate interact during the phosphoryl transfer, the relative positions of N and P are believed to change during catalysis Moreover, mutagenesis studies suggest that the A domain interacts with the P domain [5] In the crystal structure, the A domain
is a structurally independent and relatively far from the other domains, which suggests that it should also move significantly toward P during turnover
In general, a P-type ATPase is thought to operate by interconverting between states termed E1 and E2 These state conversions are driven by consecutive phosphorylation and dephosphorylation reactions The binding of a sub-strate ion to the site in the TM domain activates the ATPase [6–9] The terminal phosphate of ATP is then transferred to the invariant aspartate in the large peripheral region Subsequently, the bound substrate ion is translocated through the membrane simultaneously with the conversion
of the enzyme from the E1P into the E2P state Finally, the aspartyl phosphate is hydrolyzed and the enzyme returns to the initial state It seems that the states E1 and E2 correspond to certain relative positions of P, N and A which are structurally coupled to the ion binding site in the
TM domain However, the exact mechanism of ion translocation remains to be elucidated [10–12]
Correspondence to T Haltia, Institute of Biomedical
Sciences/Bio-chemistry, PO Box 63 (Biomedicum Helsinki, Haartmaninkatu 8),
FIN-00014, University of Helsinki, Helsinki, Finland.
Fax: + 358 9 191 25444, Tel.: + 358 9 191 25407,
E-mail: Haltia@cc.helsinki.fi
Abbreviations: TM, transmembrane; WD, Wilson disease.
*Note: These authors contributed equally to this study.
(Received 11 January 2002, accepted 23 January 2002)
Trang 2The Escherichia coli genome codes for two heavy metal
transporting P-type ATPases, named ZntA and CopB,
which are involved in the transport of zinc, lead, cadmium
and copper [13–16] Both bacterial proteins are homologous
with the Wilson disease (WD) ATPase, possessing a number
of common sequence motifs (see Fig 1) [17–19] The WD
ATPase is a copper pump More than 100 missense
mutations in 87 residues of the WD ATPase are known to
associate with the disease [20] About 30% of WD patients carry the mutation H1069Q in the motif HP [16,17,21], which appears to reside in the N domain of the protein [4]
WD is a recessively inherited hepatic and neurologic disorder, in which copper secretion to bile is defective [17,21–23] This leads to accumulation of toxic amounts of copper in the liver, kidney and the brain Mutations of the
WD ATPase can result in a spectrum of defects such as impaired copper transport by correctly localized protein, misfolding, degradation or mislocalization of the ATPase in the endoplasmic reticulum or the inability to undergo copper-dependent trafficking [24,25] The overall conse-quence is that there is no active WD ATPase molecules in the trans-Golgi network, cytoplasmic vesicles or plasma membrane, which function in copper secretion in healthy liver cells
Here we have studied ZntA, a zinc-transporting ATPase from E coli, which belongs to the same subclass of P1-type ATPases as the Wilson disease protein [26,27] In this paper,
we report characterization of three site-directed mutants P401L, D628A and P634L which mimic WD mutations P992L, D1267A and P1273L, respectively In addition, we have studied the HP motif by characterizing the mutants H475Q and P476L
M A T E R I A L S A N D M E T H O D S Mutagenesis
The ZntA gene of E coli JM 109 had been cloned by PCR and introduced into the pTrcHisA vector (Invitrogen) using the restriction sites of BamHI and KpnI [26] Site-directed mutagenesis was carried out using the overlap extension method [28,29] The primers used can be found in Table 1
To rapidly identify the clones with desired mutations, a
Fig 1 Membrane topology of ZntA The positions of the
phos-phorylation, nucleotide binding and actuator domains are shown The
locations of several well-conserved sequence motifs as well as the
residues mutated in this work are indicated Circled P connected to
D436 denotes the invariant phosphorylated aspartate in the P domain.
The motif CPX is an intramembraneous metal binding site present in
all heavy metal transporting P-type ATPases (X ¼ C,H,S) The
location of the domains and sequence motifs is based on the crystal
structure of Ca-ATPase [4], the membrane topology of which is likely
to differ from that of ZntA.
Table 1 Primers used in mutagenesis of ZntA Desired point mutations are marked in bold, the silent mutations are in italics The extra restriction site created by the silent mutation is underlined.
Mutation Primers
Restriction site(s) Created Used in cloning Pro401Leu
Fragment 1 Forward: 5¢-CAA CTG GCG TTT ATC GCG ACC ACG CT-3¢ NheI EcoNI/AgeI
Reverse: 5¢-GAG GTA ATC GCC GCT AGC GTT GAG ATA AC-3¢
Fragment 2 Forward: 5¢-GTT ATC TCA ACG CTA GCG GCG ATT ACC TC-3¢
Reverse: 5¢-CAG CGT ACC GGT TTT ATC AAA CGC CAC C-3¢
Pro476Leu
Fragment 1 Forward: 5¢-TAA AAC CGG TAC GT T AAC CGT CGG TAA ACC G-3¢ HpaI AgeI/KpnI
Reverse: 5¢-CTT GCG CCA GTA GAT GCG TCG CG-3¢
Fragment 2 Forward: 5¢-CGC GAC GCA TCT ACT GGC GCA AG-3¢
Reverse: 5¢-CCC ATA TGG TAC CCC TTA TCT CCT GCG-3¢
Asp628Ala
Fragment 1 Forward: 5¢-TAA AAC CGG TAC GT T AAC CGT CGG TAA ACC G-3¢ HpaI AgeI/KpnI
Reverse: 5¢-TCG TTA ATA CCG GCA CCG ACC ATC GCC A-3¢
Fragment 2 Forward: 5¢-TGG CGA TGG TCG GTG CCG GTA TTA ACG A-3¢
Reverse: 5¢-CCC ATA TGG TAC CCC TTA TCT CCT GCG-3¢
Pro634Leu
Fragment 1 Forward: 5¢-TAA AAC CGG TAC GT T AAC CGT CGG TAA ACC G-3¢ HpaI AgeI/KpnI
Reverse: 5¢-GGC AGC TTT CAT CGC TAG CGC GTC-3¢
Fragment 2 Forward: 5¢-GAC GCG CTA GCG ATG AAA GCT GCC-3¢
Reverse: 5¢-CCC ATA TGG TAC CCC TTA TCT CCT GCG-3¢
Trang 3silent mutation producing an extra restriction site was also
introduced All mutations were verified by sequencing the
relevant region of the expression construct Partial
charac-terization of the mutant H475Q has been reported before
[26]
Expression of His-tagged ZntA
Wild-type and mutated versions of ZntA were expressed as
His-tagged recombinant proteins in E coli TOP 10 as
described in our previous work [26] Expression of the
protein was induced with 100 lM isopropyl thio-b-D
-galactoside 1 h 45 min after inoculation Cells were
harves-ted 5 h after induction and stored at)20 °C
Isolation of the membrane fraction and determination
of the expression levels
The membrane fraction of the cells was isolated using the
protocol described before [26] The membranes were
suspended into the storage buffer (50 mM Tris, pH 8.0,
300 mM NaCl, 20% glycerol, 2 mM 2-mercaptoethanol,
0.5 mMphenylmethanesulfonyl fluoride) to a final
concen-tration of 10 mg of proteinÆmL)1 and stored at )70 °C
Protein concentration was measured with the BCA protein
assay kit (Pierce) The expression levels of the mutants were
analyzed using SDS/PAGE on 12% gels combined with
Coomassie Blue staining (30 lg membrane protein per
lane), or with Western blotting with an anti-HisG Ig
(Invitrogen) The intensitities of the Coomassie stained
bands assigned to monomeric recombinant ZntA were
quantified using AIDA software (version 2.00, Raytest
Isotopenmessgera¨te GmbH) The intensity values were used
to normalize the activity and phosphorylation
measure-ments so that the results in Figs 3–5 do not depend on the
expression levels of the mutants In the Western blotting
experiments, 10 lg of solubilized membrane protein was
analyzed on an SDS/PAGE gel and blotted on a poly
(vinylidene difluoride) membrane His-tagged proteins were
visualized using a ProtoBlot II AP system for mouse
antibodies (Promega) For N-terminal sequence analysis,
proteins separated in SDS/PAGE were electrotransferred
onto a poly(vinylidene difluoride) membrane and stained
briefly with a Coomassie Blue solution prepared without
acetic acid The 67-kDa band was identified and applied to
an Applied Biosystems 477A protein sequenator
ATPase activity measurements
The ATPase activity of the membrane fraction was
determined with the inorganic phosphate analysis method
[26,30], either in the absence of zinc, or in the presence of
20 lMZnSO4
Phosphorylation assays
Phosphorylation of the membrane fraction by [c-33P]ATP
and by 33Pi (Amersham Pharmacia) were carried out as
described previously [26], except that the total ATP
concentration was 2.5 lM and that 5 lCi of [c-33P]ATP
was used per reaction The analysis of the phosphorylated
samples on acidic 8% SDS/PAGE and imaging of the gels
by a BAS-1800 Bio-imaging analyzer (Fuji) were performed
as described previously [26] In this work only the monomer (92 kDa) and the dimer (190 kDa) bands were analyzed The exposure times of the dried gels on the imaging plate ranged from 1 to 5 h The error bars in Figs 3–7 show the standard deviation of three to four independent measure-ments
Dephosphorylation assays The rate of dephosphorylation of the phosphoenzyme intermediate was determined both in the absence of ADP (dephosphorylation via the E2P intermediate) and in the presence of ADP (dephosphorylation directly from the E1P form) The membrane fractions were first phosphorylated with [c-33P]ATP as in the phosphorylation assay above in a total volume of 680 lL (reaction to be stopped with EDTA)
or 540 lL (reaction to be stopped with EDTA and ADP) Phosphorylation was allowed to proceed for 30 s after which the reaction was stopped with 5 mM EDTA with
250 lM ADP or with 5 mM EDTA alone Samples of
160 lL were then taken at time points of 0, 5, 10, 20 s and
1 min after adding the stopper When the EDTA/ADP stopper was used, the last sample was taken at 20 s The samples were transferred to tubes containing 40 lL of cold trichloroacetic acid Due to technical reasons, the 0 s sample had actually to be taken at 10 s prior to adding the stopper However, a control experiment showed that the phosphory-lation level of the ATPase was nearly constant for 2 min if
no EDTA was added (results not shown) Samples were analyzed as in the phosphorylation assays
R E S U L T S Expression of the mutants Wild-type and mutant proteins were produced as N-terminally His-tagged recombinant proteins All mutant polypeptides are expressed at levels clearly visible in a Coomassie stained SDS/PAGE gel (Fig 2), which was routinely used to estimate the amount of ZntA protein in
Fig 2 Expression levels of the wild-type and mutant enzymes.
A standard SDS/PAGE gel stained with Coomassie blue The arrow marked with M on the right points at the ZntA monomer (80 kDa) The 67-kDa band, marked with F, is an N-terminally cleaved pro-teolytic fragment of the ATPase, persistently present despite the use of protease inhibitors The faint high molecular mass band may represent
a ZntA dimer (D) Samples were prepared from the membranes of the vector only control strain (lane 1), wild-type (lane 2), P401L (lane 3), H475Q (lane 4), P476L (lane 5), D628A (lane 6), P634L (lane 7) On each lane of a 12% SDS/PAGE gel, 30 lg of membrane protein was loaded.
Trang 4each membrane batch used for the assays described below.
The genomic ZntA gene is not expressed under our growth
conditions [26] A major band at 80 kDa represents a ZntA
monomer, whereas a minor high molecular mass band is
assigned to a ZntA dimer These assignments were verified
using a Western immunoblot with an antibody against the
His-tag (data not shown) A weaker band at 67 kDa is not
recognized by the antibody, although it is clearly observed
in the phosphorylation assays (Figs 4 and 5) N-terminal
sequencing shows that it lacks the His-tag and the first 71
residues of ZntA [The cleavage site is nine residues after the
CXXC motif (Fig 1) The cleaved protein can be
phos-phorylated by ATP and Pi While its phosphorylation by
ATP seems very similar to that of the uncleaved monomer
(Fig 4A), the cleaved fragment is more intensely
phosphor-ylated by Pi (Fig 5A) Although the behavior of the
fragment may be of interest, particularly regarding the
function of the CXXC domain, its properties are not those
of native ZntA For this reason, we have excluded the
fragment from further analysis here.]
The H475Q, D628A and P634L mutant proteins are
produced at levels similar to the wild-type The amounts of
the P401L and P476L ATPases are about 60% of the
wild-type level In assays described below, the same amount of
total protein has been used The numerical values obtained
have then been normalized to the wild-type expression level
using normalizing factors determined from a Coomassie
stained SDS/PAGE gel (see Materials and methods)
ATPase activity
The zinc-stimulated ATPase activity of bacterial
mem-branes is specific for ZntA [26] All mutants have a
zinc-dependent ATPase activity ranging from 0 to 37% of the
wild-type activity (Fig 3A), showing that the mutated
residues perform important roles in the ATPase As
mentioned above, the mutant D628A is inactive, a finding
consistent with mutagenesis studies of sarcoplasmic
Ca-ATPase and chemical modification experiments with
Na,K-ATPase [31–33] (but, see [34]) In the crystal structure
of sarcoplasmic Ca-ATPase (in the E1 state) [4], the
counterpart of D628 is D703 which resides in the proximity
of the phosphorylated aspartate D351 It may be hydrogen-bonded to another invariant aspartate (D707) in the hinge motif
The mutant P401L has about 18% of the ATPase activity left (Fig 3) Because this mutation is located nine residues toward the C-terminus from the metal binding site (the motif CPC in the sixth TM helix of ZntA in Fig 1), the low zinc dependent ATPase activity could be a consequence of decreased affinity of the metal binding site To study this possibility, we measured the ATPase activity of the wild-type and P401L ATPases in different concentrations of zinc The mutational effect cannot be compensated for by increasing the concentration of the metal ion (data not shown), suggesting that the low ATPase activity of the P401L mutant is not due to lowered affinity for zinc Phosphorylation by ATP and Pi
The phosphorylated intermediate which is formed during the transport cycle is a hallmark of all P-type ATPases In ZntA, phosphorylation by ATP is stimulated by Zn2+,
Cd2+, Pb2+ and Cu2+[26] The resulting E1P state can react with ADP to remake ATP In contrast, in the absence
of substrate ions, ZntA and other P-type ATPases can be pulled into the E2state and be phosphorylated by inorganic phosphate Pi[9] The E2P state is ADP insensitive The inactive mutant D628A is not phosphorylated at all
by ATP (Fig 4), consistent with the properties of the corresponding mutant of the sarcoplasmic Ca-ATPase [31] The mutant P401L retains 12% of the wild-type phos-phorylation by ATP, thus exhibiting a marked reduction in the formation of the ADP-sensitive phosphointermediate The mutants of the HP motif (H475Q and P476L) show ATP-driven phosphorylation levels of 32% and 26% of the wild-type, respectively In contrast, the P634L mutant has less than 10% of the ATPase activity left but nevertheless shows normal phosphorylation by ATP (Fig 4) We conclude that the mutation interferes with a catalytic step which is beyond the ATP-dependent phosphorylation reaction
The wild-type ZntA reacts with Piin the absence of zinc (Fig 5); if 30 lM Zn2+is present, phosphorylation by Pi
decreases to 20% of the maximal level (Fig 5C) All the mutants are much less reactive with Pithan the wild-type (Fig 5A,B) In particular, while the P634L mutant phorylates almost normally with ATP, hardly any phos-phorylation is observed with Pi With the mutant P401L, the remaining Pi-phosphorylation (8% of the wild-type level) is less sensitive to the presence of Zn2+than in the wild-type (Fig 5C)
Dephosphorylation kinetics
In addition to measuring the formation of aspartyl phos-phate, the catalytic cycle of a P-type ATPase can be characterized by determining the decay rate of the phos-phorylated intermediate The aspartyl-phosphate com-pound can decompose in two ways: in the normal, forward reaction along the route E1P fi E2P fi E2,
or, in the presence of extra ADP, in a reversal of the normal reaction E1P fi E1(plus ATP)
The P401L mutant dephosphorylates more slowly than the wild-type both in the absence and in the presence of
Fig 3 Normalized zinc-dependent ATPase activity of the wild-type and
mutated enzymes The ATPase activity (percentage of the activity of the
wild-type) present in membranes shown The ATPase activity present
in the absence of zinc has been subtracted The concentration of zinc
was 20 l M In each measurement, 50 lg of membrane protein was
used The error bar is the standard deviation of three to four
meas-urements.
Trang 5ADP (Fig 6) In contrast, the behavior of the P634L
enzyme is strikingly different depending on whether ADP is
present or not In the forward reaction, i.e in the absence of
added ADP, the P634L ATPase has a clearly slower
dephosphorylation rate than the wild-type (Fig 6A)
How-ever, in the presence of 250 lMADP (a hundred-fold excess
of ADP compared to ATP), the enzyme behaves like the
wild-type and is fully dephosphorylated after 5 s (Fig 6B),
which, for technical reasons, is the first time point To slow
down the dephosphorylation reaction, the concentration of
ADP was reduced to 25 lM Under these conditions, the
P634L ATPase dephosphorylates faster than the wild-type
enzyme (Fig 6C)
The dephosphorylation rates of the P476L ATPase are
slow in both assays (Fig 7) Even in the presence of excess
ADP, the dephosphorylation is considerably slower than
that of the wild-type enzyme The mutant H475Q, targeted
at the neighboring residue in the HP motif, behaves essentially the same way but the effects are slightly milder compared to the mutant P476L (Table 2)
D I S C U S S I O N
We have used here a bacterial zinc transporting P-type ATPase to study the effects of five site-specific mutations, of which four mimic mutations found in WD patients All our ZntA mutants have clear functional defects However, one
of the most common WD mutations (H1069Q, correspond-ing to H475Q in ZntA) has been shown to result in mislocalization of the WD ATPase in the endoplasmic reticulum [24] This trafficking defect as well the difficulty of producing enough of mutant protein has complicated the attempts to study the roles of the ATPase residues mutated
in WD By using a bacterial system, we have been able to overcome these problems (for an analogous study, see Bissig
et al [27])
Owing to the recent elucidation of the crystal stucture of Ca-ATPase [4], we can interpret some of the results in a structural context As deduced from the Ca-ATPase struc-ture, two of our ZntA mutants (D628A and P634L) reside
in the P domain while two (H475Q and P476L) are likely to
be in the N domain The well-conserved residue P401 is
Fig 4 Phosphorylation of the wild-type and mutant enzymes with
33 P-ATP in the presence of zinc (A) Phosphoproteins visualized in
acidic SDS/PAGE The major bands are thought to represent a ZntA
multimer, dimer (apparent molecular mass about 200 kDa), monomer
(97 kDa) and a proteolytic fragment (66 kDa), respectively
Mem-branes from the vector-only control strain do not show any bands [26].
Membranes from the wild-type (lane 1), P401L (lane 2), H475Q
(lane 3), P476L (lane 4), D628A (lane 5), P634L (lane 6) The
phos-phorylation reaction was performed on ice at pH 6.0 using 2.5 l M
ATP and a labeling time of 30 s (see Materials and methods) The
concentration of Zn 2+ was 30 l M 25 lg of membrane protein of was
loaded on each lane of an acidic 8% SDS/PAGE gel (B) Quantitation
of the intensity of monomer and dimer bands The background
phosphorylation measured with no zinc present has been subtracted.
The data has been normalized to the same concentration of ZntA.
Fig 5 Phosphorylation of the wild-type and mutant enzymes with33P i (A) Phosphorylation of the wild-type and mutant proteins in the absence of zinc wild-type (lane 1), P401L (lane 2), H475Q (lane 3), P476L (lane 4), D628A (lane 5), P634L (lane 6) The phosphorylation reaction was performed at room temperature at pH 6.0 using 100 n M
33
P i and a labeling time of 10 min On each lane of the acidic SDS/PAGE gel, 25 lg of membrane protein was loaded Membranes from the vector-only control strain do not contain any phosphorylated ZntA [26] (B) Quantitation of the intensity of monomer and dimer bands The data has been normalized as above (C) The effect of 30 l M
Zn 2+ on the P i -phosphorylation of the wild-type and the P401L enzymes (gray columns) Note that the maximal phosphorylation of the P401L mutant is only 8% of the wild-type level (see Fig 5B) White columns, no added zinc present.
Trang 6located in a sequence that connects the putative metal
binding site in the TM domain to the phosphorylation site
D436 D628A and P634L are both situated in the highly
conserved hinge motif near the C-terminal end of the P
domain Residues H475 and P476 form the so-called HP
motif, which is characteristic for heavy metal transporting
P-type ATPases A summary of the mutational effects is
presented in Table 2
The P401L mutant has some 12% of the zinc-dependent
ATPase activity left, is poorly phosphorylated by both ATP
and Piand its both dephosphorylation rates are slow The
remaining phosphorylation by Piis not as sensitive to zinc as
it is in the wild-type The mutation thus influences many
steps in the phosphorylation cycle One way to explain such
a general effect is to assume that the function of the
phosphorylation site itself is affected As indicated in Fig 1, P401 resides near the cytoplasmic end of the sixth TM helix Inspection of the Ca-ATPase structure shows that this helix (the fourth TM helix in Ca-ATPase) is partially unwound in its middle portion Assuming a similar structure for ZntA, P401 should be located in the cytoplasmic helical portion close to the membrane-aqueous interphase This helix connects the metal binding site to the P domain and its catalytic aspartate (D436 in ZntA) For this reason, a plausible interpretation of the data is that the mutant has a defect in the communication between the metal binding and phosphorylation sites It should be noted that binding of the metal ion initiates the catalytic cycle [6] This binding event
in the TM part should trigger changes in the P and N domains; these changes are prerequisites for further catalytic
Fig 7 Dephosphorylation kinetics of the wild-type (m) and the mutants H475Q (j) and P476L (r) Analysis of the dephos-phorylation rate in the absence of ADP (A) and in the presence of 250 l M ADP (B) Experimental conditions as in Fig 6.
Fig 6 Dephosphorylation kinetics of the wild-type (m) and the mutants P401L (h) and P634L (e) Phosphorylation conditions as in Fig 4 After 30
s labeling reaction with ATP, the reaction was stopped with 5 m M EDTA (A), 250 l M ADP and 5 m M EDTA (B) or 25 l M ADP and 5 m M EDTA (C) For each time point, 25 lg of membrane protein was analysed on a lane of an acidic SDS/PAGE gel.
Table 2 Summary of the mutational effects relative to the wild-type The activity values have been normalized as explained in the text.
Mutant
ATPase
activity
(%)
Phosphorylation (%) Dephosphorylation
Interpretation
by ATP by P i by ADP P401L 18 12 8 Slow Slow Communication between the metal binding site
and the P domain is affected.
H475Q 37 32 14 Slow Slow Interaction between the N and P domains is affected.
An effect on nucleotide binding is also possible P476L 23 26 11 Slow Slow As above.
D628A 0 0 3 a – – The mutant is unable to phosphorylate itself.
D628 plays a role in the catalytic site.
P634L 7 100 2b Slow Very fast The mutant is impaired in the transitions E 1 -P fi E 2 -P
and E 2 fi E 2 P.
It favours the E 1 state P634 may reside at the interface between the domains P and A.
a No clear bands Only smear observed b Weak but clear dimer and monomer bands observed.
Trang 7steps Defective allosteric communication between the metal
site and the P domain may thus result in a general defect in
the phosphorylation cycle as observed here We suggest that
P401 plays a role in coupling the metal binding and
phosphorylation sites
The counterparts of D628 and P634 in Ca-ATPase, D703
and P709, respectively, are located near the phosphorylated
D351 and constitute part of the phosphorylation site [4]
The crystal structure by Toyoshima et al [4] shows that
D703 may be hydrogen-bonded to the invariant D707 of the
hinge motif In ZntA, the mutation D628A inactivates the
ATPase Moreover, this mutant could not be
phosphoryl-ated at all in our assays, which is expected if the mutphosphoryl-ated
residue has an essential structural and/or catalytic function
in the phosphorylation site Similar results have been
reported from studies of the corresponding site-directed
mutant of the sarcoplasmic Ca-ATPase [31] However,
Pedersen et al [34] found that the analogous mutation in
the Na+/K+-ATPase yielded an enzyme with 20% of the
wild-type ATPase activity Regarding the role of D628,
several recent studies agree that this residue is involved in
binding of Mg2+[34–37] Moreover, the E1to E2transition
may be linked to changes in Mg2+ligation so that in the E1
state Mg2+is ligated by residues in the hinge motif in the P
domain and by residues in the N domain, whereas in the E2
state the latter are replaced by residues in the TGES motif of
the A domain [37] (cf [34,35]) Taken together, both
mutagenesis and structural data agree that D628 has a very
important role in the phosphorylation site of P-type
ATPases A direct catalytic function, such as assisting in a
step during the phosphoryl transfer, is possible
The P634L mutant retains less than 10% of the wild-type
ATPase activity, but phosphorylation with ATP is normal
Yet, phosphorylation with Piis almost completely lost The
phosphorylation results suggest that the mutation stabilizes
the E1 conformation, leading to the accumulation of the
species E1P in the phosphorylation experiment with ATP
This conclusion is supported by the dephosphorylation
studies in which the mutant showed a very fast
ADP-dependent dephosphorylation, whereas the normal, forward
dephosphorylation via the E2 state was slow The role of
P634 has also been studied in the case of Ca-ATPase by
substituting it with an alanine (mutant P709A [31]) This
mutant, however, showed a much milder phenotype (60%
of the wild-type ATPase activity and almost wild-type
phosphorylation properties) than our mutant which
mimicks a pathogenic WD mutation In the structure of
the Ca-ATPase in the E1state [4], P709 is solvent-exposed; it
is possible that the tolerance for the substitutions at this site
is related to the size and hydrophobicity of the substituting
side chain Inspection of a theoretical model of the
Ca-ATPase in the E2 state (PDB accession no 1FQU;
F Nakasako & C Toyoshima Tokyo University, Japan)
suggests that P709 can interact with the A domain (see also
[37]) In any event, our results support the idea that the
hinge motif GDGXNDXP is not only directly involved in
the phosphorylation reaction, but may also be important for
the E1to E2conversion
The mutants H475Q (which is analogous to one of the
most common Wilson disease mutations, H1069Q of WD
ATPase) and P476L have similar characteristics They
retain 37% and 23% of the wild-type ATPase activity,
respectively, and are poorly phosphorylated by ATP and P
Low but significant ATPase activity and phosphorylation
by ATP were also observed in the CopB mutant equivalent
to H475Q [27] Both our mutants are dephosphorylated slowly in the forward reaction (no ADP added) as well as in the ADP-dependent dephosphorylation The latter obser-vation, taken together with the likely location of the HP motif in the N domain, might indicate that these mutants have a defect in nucleotide binding In order the phos-phorylation reaction to occur, the N domain with bound ATP must interact with the P domain The mutations in the
HP motif might perturb this interaction resulting in a state intermediate between E1 and E2 This would provide an explanation for the impaired Pi phosphorylation, which requires the occupation of the E2 state The severely impaired ADP-dependent dephosphorylation indicates that also the E1 state of the mutant differs from that of the wild-type Interestingly, the nearby WD mutation homo-logue E470A results in an ATPase that prefers the E2state [26]
In conclusion, we have demonstrated that ZntA can be used to study the functional consequences of mutations that are analogous to those causing WD From the enzymolog-ical point of view, characterization of the mutants that imitate the pathogenic mutants can yield interesting func-tional data about P-type ATPases In particular, we have shown here that P634 in the hinge motif plays a role in the transition between the states E1and E2 Together with the recent determination of the crystal structure of the sarco-plasmic Ca-ATPase [4], analysis of these and other WD mutant homologues may help in further delineating the key features of these complex ion pumps
A C K N O W L E D G E M E N T S
We thank Dr Pentti Somerharju for comments on the manuscript,
Dr Marc Baumann for N-terminal sequencing and Katja Sissi, Teija Inkinen and Lea Armassalo for help with laboratory work Financial support was provided by the University of Helsinki, the Academy of Finland (program 44895), the Magnus Ehrnrooth Foundation, and the Sigrid Juselius Foundation.
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