Báo cáo Y học: Introducing Wilson disease mutations into the zinc-transporting P-type ATPase ofEscherichia coli The mutation P634L in theÔhingeÕ motif (GDGXNDXP) perturbs the formation of the E2P state pdf

Introducing Wilson disease mutations into the zinc-transporting P-type ATPase of Escherichia coli The mutation P634L in the ÔhingeÕ motif (GDGXNDXP) perturbs the formation of the E 2 P 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. T he mutants P 401L, D628A and P 634L corres- pond to the W ilson disease mutations P992L, D1267A and P1273L, respectively. The mutations D628A and P634L are located in t he 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 w hereas the m utations 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 w ild-type activity. The mutants P401L, H475Q and P476L are poorly phosphorylated by both ATP and P i . 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 P i is almost absent. In the absence of added ADP the P 634L mutant dephosphorylates much more slowly than the wild- type, whereas in the presence of A DP the dephosphorylation rate is faster tha n that of the w ild-type. We conclude th at the mutation P634L affects the conversion between the states E 1 PandE 2 P so t hat the mutant favors the E 1 or E 1 P 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 charac teristic feature i s t he presence of an aspartate i n 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 (P 1 -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 t he 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 w ith 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 a spartate 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 t hought to operate by interconverting between states termed E 1 and E 2 .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 aspar tate i n t he large p eripheral r egion. Subsequently, the bound substrate ion is translocated through the membrane simultaneously with the conversion of the e nzyme from the E 1 P into t he E 2 P state. Finally, the aspartyl phosphate is hydrolyzed and t he enzyme returns to the initial state. It seems that the states E 1 and E 2 correspond to certain relative positions o f P, N and A which a re 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) Eur. J. Biochem. 269, 1579–1586 (2002) Ó FEBS 2002 The Escherichia coli genome codes for two heavy metal transporting P-type ATPases, named ZntA and CopB, which are involved in the transport o f zinc, lead, cadmium and copper [13–16]. Both bacterial proteins are homologous with the Wilson disease (WD) ATPase, possessing a number of common sequence m otifs (see Fig. 1) [17–19]. The WD ATPase is a copper pump. More than 100 m issense mutations in 87 residues of t he 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 rece ssively i nherited 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. M utations of the WD ATPase can result in a spectrum of d efects such as impaired copper transport by correctly localized protein, misfolding, degradation or mislocalization of the ATPase in the endoplasmic r eticulum or the inability to undergo copper-dependent trafficking [24,25]. The overall conse- quence i s that there is no active WD ATPase molecules in the trans-Golgi network, cytoplasmic vesicles or p lasma 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 P 1 -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. MATERIALS AND METHODS Mutagenesis TheZntAgeneofE. 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 d omains are shown. The locations of several well-conserved sequence motifs as w ell as the residues mu tated in this work are indicated. Circled P connected to D436 denotes the invariant phosphorylated aspartate in th e 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 i s 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 sile nt 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 AC GCTA 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¢ 1580 J. Okkeri et al. (Eur. J. Biochem. 269) Ó FEBS 2002 silent mutation producing an extra restriction site was also introduced. All mutations were verified by sequencing the relevant r egion 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 i n our previous work [26]. E xpression of the protein was induced with 100 l M 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 t he cells was isolated u sing the protocol described before [26]. The membranes were suspended into t he storage buffer ( 50 m M Tris, pH 8.0, 300 m M NaCl,20%glycerol,2m M 2-mercaptoethanol, 0.5 m M phenylmethanesulfonyl fluoride) to a final concen- trationof10mgofproteinÆmL )1 andstoredat)70 °C. Protein concentration was me asured with the BCA protein assay kit (Pierce). The exp ression levels of the mutants we re analyzed using SDS/PAGE on 12% gels combined with Coomassie Blue staining (30 lg membrane protein per lane), or with Western blotting w ith an a nti-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 b lotting experiments, 10 lg of solubilized membrane protein was analyzed on an SDS/PAGE gel a nd blotted on a poly (vinylidene difluoride) membrane. His-tagged proteins were visualized using a ProtoBlot II A P 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 t o an Applied Biosystems 477A protein s equenator. 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 l M ZnSO 4 . Phosphorylation assays Phosphorylation of the membrane fraction by [c- 33 P]ATP and by 33 P i (Amersham Pharmacia) were carried out as described previously [26], except that the total ATP concentration was 2.5 l M and that 5 lCi of [c- 33 P]ATP was used per reaction. The analysis of the phosphorylated samples on acidic 8% SDS/PAGE and imaging of the g els 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 r ate of d ephosphorylation o f the p hosphoenzyme intermediate was determined both in the absence of ADP (dephosphorylation via the E 2 P intermediate) and i n the presence of ADP (dephosphorylation directly from the E 1 P form). The membrane fractions were first phosphorylated with [c- 33 P]ATP as in the phosphorylation a ssay 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 t o proceed for 30 s after which the reaction was stopped with 5 m M EDTA with 250 l M ADP or with 5 m M EDTA alone. Samples of 160 lL were then taken at time points of 0 , 5, 10, 20 s and 1 min after a dding the stopper. When the EDTA/ADP stopper was used, the last sample was taken at 20 s. The samples were tran sferred to tubes containing 40 lLofcold trichloroacetic acid. Due to technical reasons, the 0 s sample had actually to be taken at 1 0 s prior t o 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 ad ded (results not shown). Samples were analyzed as in the phosphorylation assays. RESULTS 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 i n 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 b lue. 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 p ro- 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 con trol 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 lgofmembraneproteinwas loaded. Ó FEBS 2002 Mutagenesis of ZntA (Eur. J. Biochem. 269) 1581 each 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,whereasaminorhighmolecularmassbandis assigned to a ZntA dimer. T hese assignments were verified using a W estern immunoblot with an antibody against the His-tag (data not shown). A weaker band at 67 kDa i s 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 P i . While its phosphorylation by ATP seems very similar to that of t he uncleaved monomer (Fig. 4A), the cleaved fragment is more intensely phosphor- ylated by P i (Fig. 5A). Although the behavior of the fragment may be o f interest, particularly regarding the function of the CXXC domain, its properties are not those of native ZntA. For th is reason, we have excluded t he fragment from further analysis here.] The H 475Q, D628A a nd P634L m utant proteins are produced at levels similar to the wild-type. The amounts of the P 401L and P476L ATPases are about 60% of the wild- type level. In assays described below, the same amount of total protein has been used. T he numerical values obtained have then been normalized to the wild-type expression l evel 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 a ctivity (Fig. 3A), showing t hat the mutated residues perform important roles in the ATPase. As mentioned above, the mutant D628A is inactive, a finding consistent with m utagenesis s tudies of sarcoplasmic Ca-ATPase and chemical modification experiments wi th Na,K-ATPase [31–33] (but, see [34]). In the crystal structure of sarcoplasmic Ca-ATPase (in the E 1 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 c oncentration of t he metal i on (data not shown), suggesting t hat the low A TPase activity of t he P401L mutant is not due to lowered affinity for zinc. Phosphorylation by ATP and P i The phosphorylated intermediate which is formed during the transport cycle is a hallmark of all P-type ATPases. In ZntA, phosphorylation b y ATP is stimulated by Zn 2+ , Cd 2+ ,Pb 2+ and Cu 2+ [26]. T he resulting E 1 Pstatecan 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 E 2 state and be phosphorylated by inorganic phosphate P i [9]. The E 2 P state is ADP insensitive. The inactive mutant D628A is not phosphorylated at all by ATP (Fig. 4), consistent with the properties of the corresponding mutan t of the sarcoplasmic C a-ATPase [31]. The m utant P401L retains 12% of the w ild-type phos- phorylation by ATP, thus exhibiting a m arked reduction in the formation of the ADP-sensitive phosphointermediate. The mutants of the HP motif (H475Q and P476L) show ATP-driven phosphorylation l evels of 32% and 26% of the wild-type, respectively. In contrast, the P634L mutant has less than 10% of the A TPase 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 P i in the absence of zinc (Fig. 5 ); if 30 l M Zn 2+ is present, phosphorylation by P i decreases to 20% of the maximal level (Fig. 5C). All the mutants are much less reactive with P i than the wild-type (Fig. 5A,B). In particular, while the P634L mutant phos- phorylates almost normally with ATP, hardly any phos- phorylation is observed with P i . With the mutant P401L, the remaining P i -phosphorylation (8% of the wild-type level) is less sensitive to t he presence of Zn 2+ than in the wild-type (Fig. 5 C). Dephosphorylation kinetics In addition to measuring the formation of aspartyl phos- phate, the catal ytic cycle of a P-type ATPase c an be characterized by determining the decay rate of t he phos- phorylated intermediate. The aspartyl-phosphate com- pound can decompose in two ways: in the normal, forward reaction along the route E 1 P fi E 2 P fi E 2 , or, in the presence of extra ADP, in a reversal of the normal reaction E 1 P fi E 1 (plus ATP). The P401L mutant dephosphorylates more slowly than the wild-type both in the absence a nd in the p resence of Fig. 3. Normalized zinc-dependent ATPase activity of the wild-type and mutated enzymes. TheATPaseactivity(percentageoftheactivityofthe wild-type) present in membranes shown. The ATPase activity present in the a bsence of zinc has bee n subtracted. The concentration o f zinc was 20 l M .Ineachmeasurement,50lgofmembraneproteinwas used. The error bar is the standard deviation of three to four meas- urements. 1582 J. Okkeri et al. (Eur. J. Biochem. 269) Ó FEBS 2002 ADP (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 A DP, the P634L ATPase has a clearly slower dephosphorylation r ate than the wild-type (Fig. 6A). How- ever, in the presence of 250 l M ADP (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 l M . Under these conditions, the P634L ATPase dephosphorylates faster than the wild-type enzyme (Fig. 6 C). 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 t han that of the wild-type enzyme. The mutant H475Q, targeted at the neighboring r esidue in th e HP m otif, behaves essentially the same way but the effects are slightly milder compared to the mutant P476L (Table 2). DISCUSSION We have used here a b acterial zinc transporting P-type ATPase to study the effects of five site-specific mutations, of which four mimic m utations found in WD patients. All our ZntA mutants have clear functional defects. However, one of the most common WD mutations (H1069Q, correspond- ingtoH475QinZntA)hasbeenshowntoresultin mislocalization of t he 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 c an 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 s train d o n ot sh ow 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 t ime of 30 s (see Materials an d methods). The concentrat ion 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 subt racted. The data has been n ormalized to the same concentration of ZntA. Fig. 5. Phosphorylation of the wild-type and mutant enzymes with 33 P i . (A) P hosphorylatio n 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 inten sity of monomer and d imer 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. Ó FEBS 2002 Mutagenesis of ZntA (Eur. J. Biochem. 269) 1583 located in a sequence that connects t he putative m etal 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 e ffects is presented i n Table 2. The P401L mutant has some 12% of the zinc-dependent ATPase activity left, is poorly phosphorylated by both ATP and P i and its both dephosphorylation rates are slow. The remaining phosphorylation by P i is not as sensitive to zinc as it is in the wild-type. The mutation thus influences many steps in the phosphorylation cycle. One way t o 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 th at this helix (the fourth TM helix in Ca-ATPase) is partially unwound in its middle portion. Assuming a similar s tructure for ZntA, P401 should be located in the cytoplasmic helical portion close to t he membrane-aqueous interphase. This helix connects the metal binding site to the P domain and its catalytic aspartate (D 436 in ZntA). For this rea son, a plausible i nterpretation o f 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 c atalytic Fig. 7. Dephosphorylation kinetics of t he wild-type (m) and the mutants H475Q (j) and P476L (r). Analysis of the dephos- phorylation rate in t he absence of A DP (A) and in the prese nce of 250 l M ADP (B). Experimental conditions as in F ig. 6. Fig. 6. Dephosphorylation kinetics of the w ild-type (m) and the mutants P401L (h) and P63 4L (e). Phosp horylation conditions as in Fig. 4. After 3 0 s labeling reaction with A TP, the reaction w as s topped with 5 m M EDTA (A), 250 l M ADP a nd 5 m M EDTA (B) or 25 l M ADP and 5 m M EDTA (C). For each time point, 25 lg of m embrane 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 be en 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 2 b 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. 1584 J. Okkeri et al. (Eur. J. Biochem. 269) Ó FEBS 2002 steps. Defective allosteric communication between the metal site and the P domain m ay thus result in a general defect in the phosphorylation cycle as observed here. We suggest that P401 plays a role in c oupling the me tal 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]showsthat D703 may be hydrogen-bonded to the invariant D707 of the hinge m otif. I n ZntA, the mutation D628A inactivates t he ATPase. Moreover, this mutant could not be phosphoryl- ated at all in our assays, which is expected if the mutated residue has an essential structural and/or catalytic function in the phosphorylation site. Similar results have been reported f rom 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 activit y. Regarding the r ole of D628, several recent studies agree that this residue is involved in binding of Mg 2+ [34–37]. Moreover, the E 1 to E 2 transition may be linked to changes in Mg 2+ ligation so that in the E 1 state Mg 2+ is ligated by residues in the hinge motif in the P domain and by residues in the N domain, whereas in the E 2 state the latter are replaced by residues in the TGES motif of the A domain [ 37] (cf. [34,35]). Taken t ogether, 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 i n 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 P i is almost completely lost. The phosphorylation r esults suggest that the mutation stabilizes the E 1 conformation, leading to the accumulation of the species E 1 P in the phosphorylation experiment with ATP. This conclusion is supported by t he dephosphorylation studies in which t he mutant showed a very fast A DP- dependent dephosphorylation, whereas the normal, forward dephosphorylation via the E 2 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, ho wever, 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 E 1 state [4], P709 is solvent-exposed; it is possible that the tolerance for the s ubstitutions 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 E 2 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 s upport the idea that the hinge m otif GDGXNDXP is not only directly involved in the phosphorylation reaction, but may also be important for the E 1 to E 2 conversion. The mutants H475Q (which is analogous to one of the most common W ilson disease mutations, H1069Q of WD ATPase) and P476L have similar c haracteristics. They retain 37% and 23% of the wild-type A TPase activity, respectively, and are poorly phosphorylated by ATP and P i . Low but significant ATPase activity and phosphorylation by ATP were a lso 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 w ith the likely location o f the HP motif in the N domain, might indicate that these mutants have a defect in nucleotide binding. In o rder 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 E 1 and E 2 . This would provide an explanation f or the i mpaired P i phosphorylation, which requires t he occupation of the E 2 state. The s everely impaired ADP-dependent dep hosphorylation indicates that also the E 1 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 E 2 state [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, c haracterization of t he mutants t hat 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 E 1 and E 2 . 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 d elineating the key features of these complex ion pumps. ACKNOWLEDGEMENTS We thank Dr Pentti Somerharju for comments on the manuscript, Dr Marc Baumann for N-terminal sequencing and K atja Sissi, Teija Inkinen and Lea A rmassalo for help with laboratory work. Financial support was provided by the University of Helsinki, the Academy of Finland (program 44895), the M agnus Ehrnrooth F oundation, and the Sigrid Juselius Foundation. REFERENCES 1. Skou, J.C. & Esmann, M. (1992) The Na,K-ATPase. J. Bioenerg. Biomembr. 24, 249–261. 2. Pedersen, L.P. & Carafoli, E. (1987) Ion motive ATPases. I. Ubiquity, p roperties, and significance to cell function. Trends Biochem. Sci. 12, 146–150. 3. Camakaris, J., Voskoboinik, I. & Mercer, J.F. (1999) Molecular mechanisms of copper homeostasis. Biochem. Biophys. Res. Commun. 261, 225–232. 4. Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. (2000) Crystal s tructure of the calcium pump of sarco plasmic reticulum at 2.6 A ˚ resolution. Nature 405, 647–655. 5. Clarke, D.M., Loo, T.W. & MacLennan, D.H. (1990) Functional consequences of mutations of c onserved amino acids in the be ta- strand domain of the Ca 2+ -ATPase of sarcoplasmic reticulum. J. Biol. Chem. 265, 14088–14092. 6. Voskoboinik, I., M ar., J., Strausak, D. & Camakaris, J. (2001) The regulation of catalytic activity of the Menkes copper-trans- locating P-type ATPase. Role of high a ffinity c opper-binding s ites. J. Biol. Chem. 276, 28620–28627. 7. De Meis, L. & Vianna, A.L. (1979) Energy interconvers ion by the Ca 2+ -dependent ATPase of the sarcoplasmic reticulum. Annu. Rev. Biochem. 48, 275–292. Ó FEBS 2002 Mutagenesis of ZntA (Eur. J. Biochem. 269) 1585 8. Inesi, G. (1985) Mechanism of calcium transport. Annu. Rev. Physiol. 47, 573–601. 9. Vilsen, B. (1995) Structure–function relationships in the Ca 2+ - ATPase of sarcoplasmic reticulum studied by use of the substrate analogue CrATP and site-directed mutagenesis. Comparison with the N a + ,K + -ATPase. ActaPhysiol. Scand. 154 (Suppl. 624),1–146. 10. Danko, S., Daiho, T., Yamasaki, K., Kamidochi, M., Suzuki, H. & Toyoshima, C. (2001) ADP-insensitive phosphoenzyme inter- mediate of s arcoplasmic r eticulum Ca 2+ -ATPase has a compact conformation resistant to proteinase K, V8 protease and tryp sin. FEBS Lett. 489, 277–282. 11. Stokes, D.L., Zhang, P., Toyoshima, C., Yon ekura, K., Ogawa, H ., Lewis, M.R. & Shi, D. (1998) Cryoelectron microscopy of the calcium pump from sarcoplasmic reticulum: two crystal forms reveal two different conformations. Acta Phys. Scand. 163 (Suppl. 643), 35–43. 12. Ku ¨ hlbrandt, W., Auer, M. & Scarborough, G.A. (1998) Structure of the P-type AT Pases. Curr. Opin. Struct. Biol. 8, 510–516. 13. Beard, S.J., Hashim, R., Membrillo-Hernandez, J., Hughes, M.N. & Poole, R.K. (1997) Zinc (II) tolerance in Escherichia coli K-12: evidence that the zntA gene (o732) encodes a cation transport ATPase. Mol. Microbiol. 25, 883–891. 14. Rensing, C., Mitra, B. & Rosen, B.P. (1997) The zntA gene of Escherichia coli encodes a Zn (II) -t ranslocating P-type AT Pase. Proc. Natl Acad. Sci. USA 94, 14326–14331. 15. Rensing, C., Sun, Y., M itra, B. & Rosen, B.P. (1998) Pb (II) - translocating P-type ATPases. J. Biol. Chem. 273, 32614–32617. 16. Rensing, C., Fan, B., Sharma, R., Mitra, B. & Rosen, B.P. (2000) CopA: An Esche richia coli Cu(I)-translocating P-type ATPase. Proc. Natl Acad. Sci. USA 97, 652–656. 17. Solioz, M. & Vulpe, C. (1996) CPx-type ATPases: a class of P-type ATPases that pump heavy metals. Trends Biochem. Sci. 21, 237–241. 18. Lutsenko, S. & Kaplan, J.H. ( 1995) Organization of P -type ATPases: significance of structural diversity. Biochem ist ry 34, 15607–15613. 19. Møller, J.V., J uul, B. & le Maire, M. (1996) Structural organiza- tion, ion transport, and energy transduction of P-type ATPases. Biochim. Biophys. Acta 1286, 1–51. 20. Cox, D.W. Wilson disease mutation database available from http://www.medgen.med.ualberta.ca/database.html 21. Cox, D.W. (1999) Disorders of copper transport. Br.Med.Bull. 55, 544–555. 22. Roberts, E.A. & Cox, D.W. (1998) Wilson disease. Bail liere’s Clin. Gastroenterol. 12, 237–256. 23. Sarkar, B . (1999) T reatment of Wilson and Menkes diseases. Chem. Rev. 99, 2 535–2544. 24. Payne, A.S., K elly, E.J. & Gitlin, J.D. (1998) Functional expres- sion of the W ilson disease protein reveals mislocalization and impaired coppe r-depende nt traffi ck ing o f t he co mmon H1069Q mutation. Proc. Natl Acad. Sci. USA 95, 10854–10859. 25. Forbes, J.R. & Cox, D.W. (2000) Copper-dependent trafficking of Wilson disease mutant ATP7B proteins. Hum. Mol. Genet. 9, 1927–1935. 26. Okkeri, J. & Haltia, T. (1999) Expression and mutagenesis of ZntA, a zinc-transporting P-type ATPase from Escherichia coli Biochemistry 38, 14109–14116. 27. Bissig, K.D., Wunderli-Ye, H., Duda, P.W. & Solioz, M. (2001) Structure-function analysis of purified Enterococcus hirae CopB copper ATPase: e ffect of Menkes/Wilson disease mu tation homologues. Biochem. J. 357, 217–223. 28. Higuchi, R., Krummel, B. & Saiki, R.K. (1988) A general method of in vitro preparation and specific mutagenesis of DNA frag- ments: study of protein and DNA interactions. Nucleic Acids Res. 16, 7351–7367. 29. Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. & Pease, L.R. (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77 , 51–59. 30. Lutter, R., Saraste, M., van Walraven, H.S., Runswick, M., Finel, M., Deatherage, J.F. & Walker, J.E. (1993) F 1 F 0 -ATP synthase from bovine heart mitoc hondria: developm ent of the purification of a monodisperse oligomycin-sensitive AT Pase. Biochem. J. 295, 799–806. 31. Vilsen, B., Andersen, J.P. & MacLennan, D.H. (1991) Functional consequences of alteration s to amino acids l ocated in the hinge domain of the Ca 2+ -ATPase of sarcoplasmic reticulum. J. B iol. Chem. 266, 16157–16164. 32. Lane, L.K., Feldmann, J.M., Flarsheim, C.E. & Rybczynski, C.L. (1993) Expression of rat alpha 1 Na,K-ATPase containing sub- stitutions of ÔessentialÕ amino acids in the catalytic center. J. Biol. Chem. 268, 17930–17934. 33. Ovchinnikov, Y.A., Dzhandzugazyan, K.N., Lutsenko, S.V., Mustayev, A.A. & Modyanov, N.N. (1987) Affinity modification of E 1 -form of Na + ,K + -ATPase revealed Asp-710 in the catalytic site. FEBS Lett. 217, 111–116. 34. Pedersen, P.A., Jørgensen, J.R. & Jørgensen, P.L. (2000) Importan ce o f conserved alpha -subunit segm ent 709GDGVND for Mg 2+ binding, phosphorylation, and ene rgy transdu ction in Na,K-ATPase. J. Biol. Chem. 275, 37588–37595. 35. Farley, R.A., Elquza, E., M u ¨ ller-Ehmsen, J., Kane, D.J., Nagy, A.K., Kasho, V.N. & Faller, L.D. (2001) 18 O-Exchange evidence that mutations of arginine in a signatu re sequence for P-type pumps affe ct i norganic p hosp hate b inding. Biochemistry 40, 6361– 6370. 36. Ridder, I.S. & Dijkstra, B.W. (1999) Identification of the Mg 2+ - binding site in the P-type ATPase and phosphatase m embers of the HAD (haloacid halogenase) superfamily by structural similarity to the r esponse r egulator CheY. Bioc hem. J. 339, 223–226. 37. Shin, J.M., Goldshleger, R., Munson, K.B., Sachs, G. & Karlish, S.J.D. (2001) Selective Fe 2+ -catalyzed oxidative cleavage of gastric H + ,K + -ATPase. J. Biol. Chem. 276, 4 8440–48450. 1586 J. Okkeri et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . Introducing Wilson disease mutations into the zinc-transporting P-type ATPase of Escherichia coli The mutation P634L in the ÔhingeÕ motif (GDGXNDXP) perturbs. phosphorylated by ATP during the catalytic cycle, hence the term P-type ATPases. Sequence motifs typical of the heavy metal transporting P-type ATPases (P 1 -ATPases