The Energy Transduction Mechanism of Na,K-ATPase Studied with Iron-catalyzed Oxidative Cleavage*

Rivka Goldshleger and Steven J. D. KarlishDagger

From the Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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This paper extends our recent report on specific iron-catalyzed oxidative cleavages of renal Na,K-ATPase and effects of E1 left-right-arrow  E2 conformational transitions (Goldshleger, R., and Karlish, S. J. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9596-9601). The experiments indicate that only peptide bonds close to a bound Fe2+ ion are cleaved, and provide evidence on proximity of the different cleavage positions in the native enzyme. A sequence HFIH near trans-membrane segment M3 appears to be involved in Fe2+ binding. Previously we hypothesized that E2 and E1 conformations are characterized by formation or relaxation of interactions within the alpha  subunit at or near highly conserved sequences, TGES in the minor cytoplasmic loop and CSDK, MVTGD, and VNDSPALKK in the major cytoplasmic loop. This concept has been tested by examining iron-catalyzed cleavage in both non-phosphorylated and phosphorylated conformations and effects of phosphate, vanadate, and ouabain. The results imply that both E1 left-right-arrow  E2 and E1P left-right-arrow  E2P transitions are indeed associated with formation and relaxation of interactions between cytoplasmic domains, comprising the minor loop plus N-terminal tail leading into M1 and major loop, respectively. Furthermore, it appears that either non-covalently or covalently bound phosphate bind near CSDK and MVTGD, and Mg2+ ions may bind to residues within TGES and VNDSPALKK and to bound phosphate. Thus cytoplasmic domain interactions seem to occur within or near the active site. We discuss the relationship between structural changes in the cytoplasmic domain and movements of trans-membrane segments that lead to cation transport. Presumably conformation-dependent formation and relaxation of domain interactions underlie energy transduction in all P-type pumps.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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The molecular mechanism whereby Na,K-ATPase transduces the free energy of hydrolysis of ATP into active transport of Na+ and K+ ions is unknown. We have a wealth of knowledge on transport reactions, covalent phosphorylation, E1/E2 conformational transitions, and cation occlusion, embodied in the Post-Albers kinetic mechanism (see Ref. 1). Active cation transport involves Nacyt-dependent phosphorylation from ATP, Na+ movement coupled to E1P right-arrow E2P, Kexc-activated dephosphorylation, K+ movement coupled to E2(K) right-arrow E1.

Knowledge of the structural basis for energy transduction is meager due to lack of information on molecular structure. The best structure of Na,K-ATPase at 20-25-Å resolution reveals only the overall shape of the protein and distribution of mass of alpha  and beta  subunits (2). Recent cryoelectron microscopy studies of Ca-ATPase and H-ATPase demonstrate the overall shape at 8-Å resolution, including "head", "neck," and membrane sectors, including 10 trans-membrane alpha -helical rods, most of which are tilted at an angle to the membrane (3, 4). The topological organization with 10 trans-membrane segments confirms that shown by a variety of techniques (5). Biochemical and molecular techniques are providing much information on residues involved in cation occlusion within trans-membrane segments (6), primarily M4, M5, and M6 (7-9), or ATP binding within the large cytoplasmic loop (see Ref. 5). These studies indicate the necessity for interactions between the ATP sites and cation occlusion sites. These interactions are mediated by E1 left-right-arrow  E2 conformational transitions, which have been studied extensively using proteolytic digestion, fluorescent probes, ligand binding, etc. (see Ref. 10 for a review and references). The fact that probes bound at different sites report the E1 left-right-arrow  E2 transition implies that substantial structural changes must occur. However, the nature of those changes has been largely obscure. All P-type pumps contain the conserved cytoplasmic sequences TGES in the minor loop between M2 and M3, MVTGD in the major loop, and TGDGVNDSPALKK in the so-called "hinge region" before M5 (5). Proteolytic cleavage and site-directed mutagenesis in these sequences usually stabilize E1 conformations, implying an involvement in the conformational transitions (see Refs. 5 and 8 for full references). Based on functional effects of mutations and proteolytic cleavages in the beta -strand minor cytoplasmic loop of sarcoplasmic reticulum Ca-ATPase, an interaction between the minor and major cytoplasmic loop near the phosphorylation site was proposed earlier (11). For yeast H-ATPase, mutations within the minor loop suggested a similar conclusion (12).

Recently, we have described specific iron-catalyzed or copper-catalyzed oxidative cleavage of renal Na,K-ATPase (13-15). The process seems to involve a site-specific mechanism in which peptide bonds close to the bound metals are cleaved, presumably by OH radicals generated locally by the Fenton reaction or a reactive metal-peroxyl derivative (16-19). Because more than one peptide bond can be cleaved from the same metal site, this technique provides information on interacting segments of individual subunits or neighboring subunits (unlike proteolytic cleavage). Incubation of Na,K-ATPase with Fe2+/ascorbate/H2O2 induces specific cleavage of the alpha  subunit at the cytoplasmic surface without cleaving the beta  subunit (13). Copper-catalyzed oxidative cleavage occurs at the extracellular surface and both alpha  and beta  subunits are cleaved (15).

Iron-catalyzed cleavages are very sensitive to the conformational state (13). In (E2) or E2(Rb) conformations, we observed four major and two minor fragments of the alpha  subunit. In E1 or E1Na conformations, cleavage was much slower and only one major cleavage was observed. Positions of cleavages were either identified exactly by N-terminal sequencing or, for fragments with blocked N termini, approximately using sequence-specific antibodies. In E2 conformations only, cleavages (identified by short sequences at or near the N termini) were found at 214ESE in the minor loop between M2 and M3, near 367CSDK after M4 which includes the phosphorylated Asp369, near 608MVTGD in the major loop, and at 712VNDS in the "hinge" region before M5. In either E2 or E1 conformations, a cleavage was seen near 263IATL, before M3. The observations suggested strongly that peptide bonds are cleaved with a probability depending on their proximity to a bound Fe3+ or Fe2+ ion, and thus imply that the different cleavage points are also in proximity to each other. Several cleavages lie in or near the highly conserved cytoplasmic sequences, suggesting that these sequences mediate mutual interactions. We proposed that, in E2 conformations, the minor and major loops interact near the conserved sequences while, in E1 conformations, the loops separate (13).

This paper extends our observations on iron-catalyzed cleavages in two ways. First, we have obtained further evidence for the site-specific mechanism. Second, we have looked at iron-catalyzed cleavages in both phosphorylated and non-phosphorylated conformations and effects of inhibitors. The results provide novel information on the energy transduction mechanism.

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Na,K-ATPase, with specific activities of 12-17 units/mg protein, was prepared from pig kidney (20) and was stored at -70 °C in a solution of 250 mM sucrose, 25 mM histidine, pH 7.2, and 1 mM EDTA (Tris). Rat axolemma microsomes (2-3 units/mg enzyme) prepared as in Ref. 21 or rat kidney microsomes (3-4 units/mg protein) were prepared as in Ref. 20. Prior to incubation with Fe2+/ascorbate/H2O2, the microsomal membrane preparations (1.5 mg/ml) were pretreated with sodium deoxycholate 1.2 mg/ml for 15 min at 20 °C, washed twice, and resuspended in a solution containing 10 mM Tris·HCl, pH 7.2.

Cleavage Reactions-- Suspensions of pig kidney Na,K-ATPase (0.1-1 mg/ml) or rat microsomal preparations (1 mg/ml) were incubated at 20 °C with freshly prepared solutions of 5 mM ascorbate (Tris) plus 5 mM H2O2, without or with added FeSO4. To arrest the reaction, 5-10 mM EDTA or 5-fold concentrated gel sample buffer with 5 mM EDTA was added. Samples were assayed for Na,K-ATPase activity or applied to gels, respectively.

Gel Electrophoresis, Blotting to Polyvinylidene Difluoride (PVDF)1, Immunoblots, Sequencing-- Procedures for running of 7.5% Tricine SDS-PAGE, including precautions prior to sequencing, electroblotting to PVDF paper, immunoblots, and microsequencing of fragments have been described in detail (22, 23). Anti-K1012-Y1016, referred to as "anti-KETYY," was used to detect fragments of the alpha  subunit. Immunoblots were stained with diaminobenzidine with metal ion enhancement (15-20 µg of protein/lane) (24) or developed by enhanced chemiluminescence (ECL; 3-5 µg of protein/lane) using anti-rabbit IgG horseradish peroxidase conjugate and the protocol supplied with ECL reagents from the 1998 Amersham Pharmacia Biotech catalogue. For quantification of bands, the stained PVDF paper or developed x-ray films were scanned with a Bio-Rad GS-690 imaging densitometer and analyzed with Bio-Rad Multi-Analyst software (version 1.01). For quantification of Coomassie-stained alpha  subunit the band was cut out of the gel, and optical density of Coomassie stain extracted into 1% SDS solution was measured at 595 nm (13).

Calculations-- Non-linear curve-fitting was performed using Enzfitter (Elsevier-Biosoft).

Materials-- For SDS-PAGE, all reagents were electrophoresis-grade from Bio-Rad. Tris (ultra pure) was from Bio-Lab, Jerusalem. L-(+)-ascorbic acid (catalogue no. 100127), 30% H2O2 (catalogue no. 822287), and alpha -chymotrypsin (catalogue no. 2307) were from Merck. Phosphocreatine, P6502, creatine phosphokinase P3755. and oligomycin O4876 were from Sigma. All other reagents were of analytical grade.

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Properties of Fe2+ Activation of Cleavage; Dependence on E1/E2 Conformations-- Fig. 1 presents an immunoblot using anti-KETYY to detect fragments produced in media containing different proportions of K+ and Na+ ions (sum 150 mM). In 150 mM K+ (E2(K), left), we observed five major fragments referred to as: a, near M1; b, at 214ESE; c, near 283HFIH (previously referred to as near IATL, see below); d, near 608MVTGD; and e, at 712VNDS. Apparent Mr values are 91.3, 80.6, 73.4, 38.2, and 26.3 kDa, respectively. By comparison with previous experiments done in low ionic strength media (13), the cleavage near 367CSDK was largely suppressed, while that near M1 was more prominent. About half the alpha  subunit was cleaved in these conditions. As K+ was replaced by Na+ ions, cleavage at ESE, near MVTGD, and at VNDS was progressively suppressed, essentially completely at 150 mM Na+ (E1Na, right), while in parallel, the cleavages near M1 and HFIH were amplified. A similar change of pattern was observed upon transfer of the enzyme from low ionic strength (10 mM Tris, pH 7, E2) to high ionic strength media (10 mM Tris, pH 7, 300 mM choline chloride, E1).


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Fig. 1.   Iron-catalyzed cleavage of the alpha  subunit at different K+ and Na+ concentrations. Na,K-ATPase (1 mg/ml) was suspended in a medium containing 10 mM Tris·HCl, pH 7.2, and 150 mM of KCl plus NaCl in the indicated proportions. 10 µM FeSO4 was added and then 5 mM ascorbate and 5 mM H2O2; after 2 min of incubation at 20 °C, the reaction was stopped with concentrated gel buffer containing 5 mM EDTA and samples were applied to the gel.

Bound Fe2+ ion is predicted to be in contact with more residues in E2 than in E1 forms (see Ref. 13 and model in Fig. 11), and thus the Fe2+ should bind more tightly in E2 forms. Fig. 2 depicts the relationship between Fe2+ ion concentration and cleavage of the alpha  subunit and inactivation of Na,K-ATPase activity, in Rb+- and Na+-containing media (E2(Rb) or E1Na, respectively). The curves are fitted well by simple hyperbolas with K0.5 values of 0.57 and 3.49 µM for cleavage of the alpha  subunit and 0.20 and 1.16 µM for inactivation of Na,K-ATPase, respectively. As discussed previously (13, 15), the 2-3-fold lower K0.5 value for inactivation of Na,K-ATPase compared with cleavage could imply that oxidative reactions occur in addition to chain cleavage and inactivate the enzyme. In any event, the 5-6-fold lower values of K0.5 in the E2(Rb) compared with the E1Na conformation, by either measure, are compatible with the prediction.


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Fig. 2.   Fe2+ concentration dependence of cleavage of the alpha  subunit and inactivation of Na,K-ATPase activity in Rb- or Na+-containing media. Na,K-ATPase (0.1 mg/ml) suspended in a medium containing 10 mM Tris·HCl, pH 7.2, and 30 mM RbCl or NaCl, and was incubated with Fe2+ at varying concentrations and 5 mM ascorbate and H2O2 for 2 min at 20 °C. Samples were either taken for determination of the Na,K-ATPase activity or applied to a gel (45 µg of protein/lane), and the alpha  subunit was quantified as described under "Experimental Procedures."

Fe2+ ions can replace Mg2+ ions in catalysis of Na-dependent phosphorylation (25) and thus the question arose whether Fe2+ ions bind to the Mg2+ site and catalyze cleavage from this site. Fig. 3 depicts the cleavages at three concentrations of Fe2+ (0, 1.5, and 15 µM added in addition to the 0.05 µM contaminant in the solutions), and Mg2+ from 0 to 7 mM. As the Mg2+ ion concentration was raised, the cleavages at ESE, near CSDK, near MVTGD, and at VNDS were progressively suppressed, while cleavages near M1 and near HFIH were amplified (Fig. 3A). Thus, Mg2+ ions stabilized an E1 conformation, noticeable especially at 7 mM. However, quantification of amounts of the fragments, by scanning the immunoblots, do not reveal any systematic influence of Fe2+ concentration on effects of Mg2+ ions, as seen in the examples in Fig. 3B. Hence, Fe2+ and Mg2+ ions do not compete and must bind at different sites.


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Fig. 3.   Iron-catalyzed cleavage of the alpha  subunit at different Fe2+ and Mg2+ concentrations. A, Na,K-ATPase (1 mg/ml) was suspended in a medium containing 10 mM Tris·HCl, pH 7.2, and MgCl2 (0, 0.15, 1.5, and 7 mM, respectively). Each of the four enzyme suspensions were divided into three aliquots, and 0, 1.5, or 15 µM FeSO4 was added together with 5 mM ascorbate/H2O2. The incubation time without or with added Fe2+ was 15 or 2 min respectively. B, diaminobenzidine-stained PVDF paper was scanned and analyzed as described under "Experimental Procedures." The figures depict the quantities of the indicated fragments at the different Mg2+ concentration as a percentage of the quantity of the fragment in the absence of Mg2+ ions.

In Ref. 13, we speculated that histidine residues in the sequence HFIH near M3 are involved in Fe2+ binding. To test this hypothesis, and also the site-specific mechanism, we compared cleavage of rat axolemma and rat kidney enzymes. Rat axolemma enzyme consists of about 65% alpha 3, 25% alpha 2, and only 10% alpha 1 isoforms, while kidney enzyme is essentially all alpha 1 isoform (26-28). In alpha 3 and alpha 2 isoforms, the second histidine is replaced by a glutamine, i.e. HFIQ. The experiment in Fig. 4A compared the time course of cleavage of axolemma and kidney enzymes. For axolemma enzyme, the cleavage near HFIH was largely absent and that at VNDS was less prominent, while the cleavages at ESE and near MVTGD were similar to those of the kidney enzyme. For axolemma, a small amount of the fragment with N terminus near CSDK also appeared, although this is less certain because the control itself contains a minor fragment with the same mobility. (The axolemma fragments cleaved near MVTGD and at VNDS have slightly lower mobility than equivalent fragments of kidney (41.8 versus 39.1 and 29 versus 27 kDa, respectively). However, this cannot be taken to indicate that the cleavage sites are different, since intact alpha 3 and alpha 2 also have a slightly lower mobility than the alpha 1 isoform, even though the Mr value of rat alpha 1 (112, 566) is slightly higher than alpha 2 (111, 580) or alpha 3 (111, 735) (27, 29).) Fig. 4B depicts cleavage of axolemma enzyme in Rb+- or Na+-containing media at different concentrations of added Fe2+ ions. The two major fragments, with N termini ESE and near MVTGD, increased in amount as Fe2+ was raised progressively to 10 µM, as did the minor fragments. In parallel experiments with axolemma and kidney enzyme (data not shown), it was found that cleavage of axolemma enzyme required significantly higher concentrations of added Fe2+ ions. For example, the K0.5 values for appearance of the fragment with N terminus near MVTGD were 1 ± 0.2 µM for kidney and 1.95 ± 0.2 µM for axolemma, respectively. In the sodium-containing medium, cleavage of both the axolemma and kidney enzymes was suppressed and the fragment with N terminus ESE and the fragment near MVTGD were not observed. In summary, axolemma and kidney enzymes differ in specificity of the cleavages and affinity for Fe2+ ions, whereas the E2(Rb) right-arrow E1Na transition affects cleavage essentially similarly.


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Fig. 4.   Iron-catalyzed cleavage of rat axolemma and kidney Na,K-ATPase. A, deoxycholate-treated rat axolemma (2.1 units/mg) or kidney (4.3 units/mg) microsomes were suspended at 1 mg/ml in a medium containing 10 mM Tris·HCl, pH 7.2, and 30 mM RbCl and incubated for the indicated times with Fe2+ 0.15 µM, plus 5 mM ascorbate/H2O2. The axolemma or kidney microsomes were applied to a gel (25 or 12.5 µg of protein/lane respectively, in order to make equal the amount of alpha  subunit in each case). B, deoxycholate-treated axolemma microsomes (1 mg/ml), suspended in media containing 30 mM RbCl or NaCl, were incubated with the indicated concentrations of FeSO4 and 5 mM ascorbate/H2O2 for 5 min. 25 µg of protein was applied per lane.

Effects of Phosphorylation, Inorganic Phosphate, Vanadate, and Ouabain-- Fig. 5 depicts iron-catalyzed cleavage of the pig kidney Na,K-ATPase in conditions of Na-dependent phosphorylation from ATP. A low concentration of ATP (5 µM) was used together with a regenerating system. In the presence of 140 mM Na+ ions, 0.5 mM Mg2+ ions, and the regenerating system, we observed cleavages typical for the E1Na form (N termini near M1 and HFIH). Upon addition of ATP and oligomycin, the pump should be phosphorylated and, due to block of the E1P right-arrow E2P conformational transition (30), the predominant form should be E1P. This condition indeed produced cleavages typical of an E1 conformation. In the absence of oligomycin, the combination of ATP/Na+/Mg2+ phosphorylates the pump, but the predominant form should now be E2P. In this condition, we observed two additional cleavages typical of an E2 form (N termini ESE and VNDS), but, strikingly, the prominent cleavage near MVTGD normally seen for unphosphorylated E2 or E2(K) forms did not appear. The cleavage near CSDK was not seen, but this was expected in the high ionic strength medium. Addition of a low concentration of Rb+ (2 mM) to the medium containing ATP/Na+/Mg2+ accelerates dephosphorylation from high affinity extracellular sites, leading to E2(Rb) as the predominant form. In this condition, the fragments with N termini ESE and VNDS were somewhat amplified, but again the cleavage near MVTGD did not appear. The latter result was surprising because the cleavage near MVTGD is very prominent in the E2(Rb) form generated directly by adding Rb+ (K+) ions to the enzyme (see Ref. 13 and this paper). The cleavage pattern in the medium containing 2 mM Rb+, 150 mM Na+, and 0.5 mM Mg2+, without ATP, shows that the enzyme remained in the E1 form. Thus, suppression of the cleavage near MVTGD in the presence of Rb+ ions and ATP/Na+/Mg2+ was associated with formation of the E2(Rb) form via dephosphorylation of E2P. It seemed possible that ATP itself or the products of its hydrolysis, ADP and Pi, were responsible for suppressing the cleavage near MVTGD, and thus a number of control experiments were performed. In low ionic strength media (E2), the cleavage near MVTGD was prominent, but neither ATP nor ADP at 5 µM had any effect (data not shown).


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Fig. 5.   Iron-catalyzed cleavage of the alpha  subunit in phosphorylated and dephosphorylated conformations. Na,K-ATPase (0.6 mg/ml) was suspended at 20 °C in a medium containing 10 mM Tris·HCl, pH 7.2, 140 mM NaCl, 2 mM creatine phosphate (sodium salt), creatine phosphokinase (48 units/ml) and where indicated 0.5 mM MgCl2, 5 µM ATP(Tris), 150 µg/ml oligomycin, and 2 mM RbCl. The samples were then incubated with 5 µM FeSO4 and 5 mM ascorbate/H2O2 for 2 min at 20 °C.

We calculated that, during incubation with ATP/Mg2+/Na+/Rb+ in Fig. 5, 200-250 µM Pi could accumulate. Therefore, we looked for effects on cleavages of Pi(Tris), 0.1-1 mM, without or with Mg2+ and Rb+ ions (Fig. 6). Addition of Pi to a medium of low ionic strength indeed suppressed the cleavages near CSDK and MVTGD, without significantly affecting other cleavages (Fig. 6A). In this condition, one could expect the enzyme to be in an E2·P form, with Pi bound non-covalently. In combination with 1 mM Mg2+ ions, addition of 2 mM Pi also partially suppressed the cleavages at ESE and VNDS (Fig. 6B, see legend for quantification based on scans). When added alone, Mg2+ ions at 1 mM or lower concentrations had little or no effect (see also Fig. 3). The presence of 1 mM Rb+ ions did not alter the effect of Pi but prevented partial suppression of cleavages at ESE and VNDS by the combination of Pi and Mg2+ ions (Fig. 6C, see legend for quantification). Another effect of the combination of Pi and Mg2+ ions was observed in experiments that examined Pi concentration dependence of suppression of the cleavage near MVTGD at 0-1 mM Mg2+. Data from scans of gels (Fig. 7) show that lower concentrations of Pi were required in the presence of Mg2+ ions. In the presence of 1 mM Mg2+ and 2 mM Pi, a substantial fraction of the enzyme should be phosphorylated as a form referred to as E2i-P, which is insensitive to Rb+(K+) ions (31). In the presence of Mg2+, Pi, and Rb+, a major fraction should not be phosphorylated (E2(Rb)·Pi) and a minor faction could be phosphorylated in a Rb+(K+)-bound form (E2-P·Rb) (31). An economical explanation of the results in Figs. 5-7 is that either non-covalently bound phosphate or covalently bound phosphate, derived from ATP or Pi, directly interfere with the cleavages near CSDK and MVTGD. In addition the cleavages at ESE and VNDS are somewhat suppressed in the phosphorylated forms (see "Discussion" and Fig. 11).


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Fig. 6.   Effects of Pi, Mg2+, and Rb+ ions on iron-catalyzed cleavage of the alpha  subunit. Na,K-ATPase (1 mg/ml) was suspended in a medium containing 5 mM Tris·HCl, pH 7.2, and 0, 0.1 or 1 mM Pi(Tris) (A) or 1 mM MgCl2 and 2 mM Pi as indicated in B or 1 mM MgCl2, 2 mM Pi, and 1 mM RbCl as indicated in C, and incubated with 5 µM FeSO4 and 5 mM ascorbate/H2O2 for 2 min at 20 °C. Amounts of fragments in arbitrary units based on scans of immunoblots: B, ESE: control, 7.2; Mg, 7.0; Pi, 6.0; Pi/Mg, 4.9; VNDS: control, 6.26; Mg, 6.0; Pi, 6.7;Pi/Mg, 4.4. C, ESE: control, 7.2; Mg, 7.3; Pi, 7.1; Pi/Mg, 5.9; VNDS: control, 6; Mg, 5.9; Pi, 6.3; Pi/Mg, 6.2.


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Fig. 7.   Co-operative effect of Pi and Mg2+ ions in suppression of the specific cleavage near MVTGD. Na,K-ATPase (1 mg/ml) was suspended in a medium containing 10 mM Tris·HCl, pH 7.2, and 0, 0.1, 1, or 5 mM Pi(Tris) and 0, 0.1, or 1 mM MgCl2. The ionic strength was maintained constant by addition of choline chloride as necessary. The suspension was incubated with 5 µM FeSO4 and 5 mM ascorbate/H2O2 for 2 min at 20 °C. The diaminobenzidine-stained PVDF paper was scanned and analyzed as described under "Experimental Procedures." The figure depicts the quantity of the fragment near MVTGD at different Pi concentrations as a percentage of that without Pi, at the MgCl2 concentration of 0, 0.1, and 1 mM.

Figs. 8 and 9 present some paradoxical observations. As seen in Fig. 8, the presence of Mg2+/ouabain somewhat suppressed cleavages near MVTGD and at VNDS, while in the presence of Pi/Mg2+/ouabain all cleavages were suppressed except the two near M1 and HFIH. Similarly, with vanadate (2 µM)/Mg2+, only the two cleavages near M1 and HFIH were observed. Thus, the cleavage pattern with Pi/Mg2+/ouabain and vanadate/Mg2+ was characteristic of E1 forms, although the enzyme should be in an E2 form in both conditions (see also Fig. 10). Fig. 9 presents another surprising finding with Pi. With the usual order of addition of the components, first equilibration of enzyme with Pi and then incubation with Fe2+/ascorbate/H2O2, the effect of Pi was as described in Fig. 6A. However, if the enzyme was preincubated with both Pi and Fe2+ prior to addition of ascorbate/H2O2, the cleavages at ESE and VNDS were also largely suppressed, although those near M1 and HFIH were largely unchanged. The development of this effect is seen in Fig. 9. After 20 min of preincubation of Pi with Fe2+, prior to addition of ascorbate/H2O2, the cleavage pattern was similar to that with Pi/Mg2+/ouabain or vanadate/Mg2+.


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Fig. 8.   Effect of Pi/Mg2+/ouabain or vanadate/Mg2+ on iron-catalyzed cleavage of the alpha  subunit. Na,K-ATPase (1 mg/ml) was suspended in a medium containing 5 mM Tris·HCl, pH 7.2, 1 mM MgCl2, and 2 mM Pi and 1 mM ouabain as indicated (A), or 0.5 mM MgCl2 and 2 µM vanadate (Tris) as indicated (B), and incubated with 10 µM FeSO4 and 5 mM ascorbate/H2O2 for 1 min at 20 °C.


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Fig. 9.   Effect of preincubation of Pi with Fe2+ ions on iron-catalyzed cleavage of the alpha  subunit. Na,K-ATPase (1 mg/ml) was suspended in a medium containing 10 mM Tris·HCl, pH 7.2, 1 mM Pi (Tris), and 5 µM FeSO4 and was incubated for 0-20 min. Then 5 mM ascorbate/H2O2 was added and the suspension incubated for 2 min at 20 °C.


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Fig. 10.   Chymotryptic digestion of Na,K-ATPase. Na,K-ATPase (1 mg/ml) was suspended in a medium containing 5 mM Tris·HCl, pH 7.2, 20 mM NaCl, or 20 mM RbCl or 7 mM MgCl2 (A) or 1 mM Pi, 1 mM MgCl2, and 1 mM ouabain (B). alpha -Chymotrypsin was added at a ratio of 1:20 (w/w) with respect to Na,K-ATPase and incubated together at 37 °C for 10 min. 150 mM RbCl was added and then 1 mM PMSF, and the mixture was incubated for 15 min at room temperature before centrifugation to remove the chymotrypsin, and solubilization of the pellet in the gel buffer. In A, 25 µg of protein were applied per lane (diaminobenzidine stain), and, in B, 5 µg were applied per lane (ECL).

Comparison of Chymotryptic and Iron-catalyzed Cleavages-- The paradoxical results just presented led us to inquire whether the expected conformations were indeed being stabilized in the different conditions Well characterized chymotryptic cleavages (32) showed that this was the case (Fig. 10). Thus, chymotryptic digestion in the Na+-containing medium (E1Na) or in a medium containing 7 mM Mg2+ ions (E1Mg) produced the prominent fragment "a," 74.6 kDa, while in the Rb+-containing medium (E2(Rb)) "a" was suppressed and "b" and "c" were more prominent. Again, as expected in the medium containing ouabain/Mg/Pi (E2-P·ouabain), "b" and "c" were major and "a" was minor. An incidental benefit was that defined chymotryptic fragments allowed better determination of the positions of two iron-catalyzed cleavage fragments, which could not be sequenced due to blocked N termini. The N terminus of fragment "a" is Ala267 (33), and sequencing of fragments "b" and "c" showed that the N termini are Val440 and Ala591, respectively. The apparent Mr values of "a" and "c" 74.6 and 39.3 kDa are 1.2 and 1.1 kDa greater than those of the closest iron-catalyzed cleavage fragments, 73.4 and 38.2 kDa respectively. Therefore, the N termini of the latter are approximately 10 residues downstream, i.e. they are near 283HFIH and 608MVTGD.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Fig. 11 depicts schematically the proposed arrangement of the peptide sequences around the bound Fe2+ ion in different states. Table I summarizes the specificity of the different cleavages and also gives a rough measure of their prominence, which reflects presumably proximity to bound Fe2+ ions.


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Fig. 11.   Schematic drawings of interactions between bound Fe2+ ions and the Na,K-ATPase in different conformational states.

                              
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Table I
Fragments observed in different conformational states
High µ, high ionic strength; low µ, low ionic strength; ***, major; **, intermediate; *, minor.

The Fe2+ Binding Site-- Previously, we proposed the site-specific mechanism in which each alpha  subunit is cleaved at only one of the different points of contact between the polypeptide chain and a bound Fe2+ ion, as depicted in Fig. 11 (13, 14). The additional observations discussed here support this notion and essentially exclude the possibility that cleavages are catalyzed by Fe2+ bound at several sites. One might consider a hypothesis that cleavages occur at two Fe2+ sites, one of which includes the points near M1 and HFIH and does not change in E1 and E2 forms, while the other includes the points at ESE, near CSDK, near MVTGD and at VNDS and exists only in E2 forms. Although it is difficult to rigorously exclude this idea, the following arguments favor one Fe2+ site.

1) Parallel suppression of cleavages at ESE, near MVTGD and at VNDS and amplification of cleavages near M1 and HFIH, upon transition from E2(K) to E1Na (Fig. 1) or other E1 forms (Fig. 3), is explained most simply by assuming that the contacts at ESE, near MVTGD and at VNDS move away while those near M1 and HFIH remain near the bound Fe2+ ion. Accordingly, the probability of cleaving either decreases or increases respectively.

2) In Fig. 2, the curves fit well to simple hyperbolae with higher apparent affinities for inactivation of Na,K-ATPase or cleavage in a Rb+-containing as opposed to a Na+-containing medium (K0.5 of 0.2 or 0.57 versus 1.16 or 3.49 µM, respectively), consistent with Fe2+ binding to a single site. Apparent free energies of Fe2+ binding, calculated from the K0.5 for inactivation or cleavage, in Rb+- compared with Na+-containing media, are -9.02 or -8.43 versus -8.01 or -7.36 kcal/mol, respectively, giving a difference of about -1 kcal/mol by either measure. Assuming that the different cleavages between the E2(Rb) and E1Na conformations are catalyzed at a separate Fe2+ site, the K0.5 for Fe2+ at this site would correspond to the -1 kcal/mol or 0.18 M, i.e. an unrealistic value that argues strongly against the idea of two sites. In contrast, the data imply that most of the binding energy for Fe2+ comes from residues that bind in either E2 or E1 forms (e.g. HFIH), while the extra contacts in E2 forms are energetically weak.2

3) Lack of competition between Mg2+ and Fe2+ ions (Fig. 3) shows that Mg2+ and Fe2+ bind at different sites. Other data discussed below (see also Fig. 11) suggest that, in the presence of phosphate, Mg2+ ions can interact with the protein near the cleavage sites at ESE and VNDS. These observations also favor the notion of one Fe2+ site rather than two separate sites, neither of which recognize Mg2+ ions.

4) Although there are significant differences in sequences of the rat alpha 3, alpha 2, and alpha 1 isoforms (85-86% identity; Ref. 29), it is likely that the different cleavage patterns of axolemma (mainly alpha 3 and alpha 2) and kidney (alpha 1) enzyme (Fig. 3) are attributable to the His286 right-arrow Gln substitution. First, the cleavage near HFIH is essentially absent in axolemma enzyme. Second, the fact that cleavages, at ESE and near MVTGD, occur in both axolemma and kidney enzymes and undergo a similar response to the E2(Rb) right-arrow E1 transition (Fig. 4B) indicates that spatial organization of the alpha 3 and alpha 1 isoforms is similar. Thus, reduced cleavage at VNDS in addition to that at HFIH suggests that the Gln for His substitution alters the disposition of both VNDS and HFIH next to the bound Fe2+ ions and weakens Fe2+ binding. Conversely, reduced cleavage at VNDS catalyzed at a separate Fe2+ site is a less likely possibility since the sequences of alpha 1, alpha 2, and alpha 3 isoforms are identical over a long stretch either side of VNDS. Unequivocal evidence for involvement of histidine residues will require site-directed mutagenesis.

5) Treatment of renal Na,K-ATPase with the histidine-specific reagent diethylpyrocarbonate prevents all iron-catalyzed cleavages, clearly implicating histidine residues.3 Histidines are found only at the cleavage site near HFIH and MVTGDH.

Effects of Phosphorylation, Inorganic Phosphate, Vanadate, and Ouabain-- Two salient features in Fig. 5 are as follows: 1) in E1P (ATP/Na+/Mg2+/oligomycin), the cleavage pattern is typical for E1; 2) in E2P (ATP/Na+/Mg2+), two cleavages typical of E2 or E2(Rb) appear (at ESE and VNDS), if less prominently than in the non-phosphorylated form (ATP/Na+/Mg2+/Rb+), but the major cleavage near MVTGD is not observed. Fig. 6 clarifies the difference between the phosphorylated or unphosphorylated E2 forms by showing the following. 1) Non-covalent binding of Pi in E2·P selectively suppresses the cleavages near CSDK and MVTGD (Fig. 6A). An equivalent form is achieved in the presence of Pi/Mg2+/Rb+ (Fig. 6C), or ATP/Na+/Mg2+/Rb+ due to ATP hydrolysis (Fig. 5). 2) In E2i-P (Pi, Mg2+), the pattern is the same as in E2-P (Fig. 6B). E2P and E2i-P represent different subconformations, as indicated by different responses to alkali cations, methyl hydroxylamine, and vanadate (31, 34, 35). Since their iron-cleavage pattern is the same, presumably the difference between E2P and E2i-P is restricted to the microenvironment of the bound phosphate. On the basis of Figs. 5 and 6, we propose that either non-covalently bound (E2·P shown in Fig. 11) or covalently bound phosphate (E2-P, E2i-P) interacts directly with residues near CSDK and MVTGD, hindering access of the bound Fe2+ and cleavage.

Mg2+ ions are tightly bound to the phosphoenzyme formed from either ATP (E2-P) or Pi (E2i-P) (31, 25, 36). In order to explain the less prominent cleavages at ESE and VNDS in the E2-P·Mg complex compared with non-phosphorylated forms (seen in both Figs. 5 and 6), and the synergism between Pi and Mg2+ ions in suppressing cleavage near MVTGD (Fig. 7), we propose that Mg2+ ions are bound near ESE and VNDS and also to the bound phosphate. In this way, access of the Fe2+ to the sites at ESE and VNDS may be reduced. In the presence of Pi, Mg2+ ions, and ouabain (Fig. 8), ouabain may induce the Mg2+ ions in the E2-P·Mg complex to bind to its contact residues more tightly or in such a way as to completely prevent access of the bound Fe2+ ions and cleavage at ESE, near CSDK and MVTGD, and at VNDS (Fig. 11). Similarly vanadate may bind near CSDK and MVTGD with Mg2+ ions bound near ESE and VNDS and to the vanadate itself, suppressing cleavages at ESE, near CSDK and MVTGD, and at VNDS (Fig. 11). Thus, the paradox that Pi/Mg2+/ouabain or vanadate/Mg2+ give E1-like cleavage patterns, although these ligands stabilize E2 forms, is explained by assuming that the bound ligands directly hinder access of the bound Fe2+ ions to the cleavage sites. Finally, the paradox (Fig. 9) that preincubation of Pi with Fe2+ leads to a pattern similar to that with Pi/Mg2+/ouabain or vanadate/Mg2+ may imply that, Fe2+ ions slowly gain access to normal Mg2+ binding sites (see Ref. 25) to produce a complex with the Pi. In this complex with Pi, the Fe2+ is itself redox-inactive, but access of the redox active Fe2+ bound near HFIH (Fig. 11) to the cleavage sites is hindered.

Although our findings do not identify precisely co-ordinating residues for Mg2+ ions and phosphate, some reasonable suggestions can be made. Based on a structural analogy to EF hand proteins and cation binding to synthetic peptides, it was proposed that Mg2+ ions are co-ordinated with Asp707 in the sequence EITAMTGDVN707DSPALKK of Ca-ATPase. (37). Studies of O18 exchange between Pi and water after mutagenesis of Asp586 of Na,K-ATPase, and sequence and structural homologies with adenylate kinase, suggest that conserved sequences 586DPPR and 608MVTGDHPITAK may be involved in co-ordination of Mg2+ ions and the gamma -phosphate group of ATP (38, 39). Proximity of the gamma -phosphate of ATP to the sequence VNDS can be inferred from covalent labeling at Asp710 (40) and at Lys719 (41). Mg2+ ions could be co-ordinated to several residues, such as Asp714 and Glu214 at VNDS and ESE as depicted in Fig. 11. Non-covalently bound phosphate could interact with Lys370 in CSDK, and both non-covalently and covalently bound phosphate could also interact with Lys618 near MVTGD (or Arg589 in the DPPR sequence).

The effect of Pi alone (Fig. 6) and synergism between Pi and Mg2+ (Fig. 7) indicate that Pi and Mg2+ bind in an unordered but positively co-operative fashion (see also Refs. 36 and 42). Our findings do not agree with a proposal that Pi binds after Mg2+ ions (38).

Implications for the Energy Transduction Mechanism-- By comparison with our initial study of iron-catalyzed cleavages, several additional conclusions emerge.

1) Characterization of E2(K) and E1Na forms by formation or relaxation of interactions between minor and major cytoplasmic loops can be modified slightly to include a role of the N-terminal cytoplasmic segment. Parallel behavior of the cleavages near M1 and M3 (HFIH) implies proximity between M1 and M3 and interactions between the segment leading into M1 and loop between M2 and M3 (probably via salt bridges; Ref. 33). Proteolytic cleavage or truncated mutants in the N-terminal segment shift the conformational equilibrium toward E1 (33, 43), as do proteolytic cleavages or mutations in the loop between M2 an M3 (Refs. 32 and 44; see Ref. 5 for references to other P-type pumps). Recently, an interaction between the cytoplasmic tail and the loop between M2 and M3 and possibly with the large cytoplasmic loop (45) has been inferred on the basis of strong synergism in the functional effects of a double mutant (with a truncation at residue 32 (a1M32) and also G233K or G233Q mutation, referred to as a1M32G233K or a1M32G233KQ). Our results fit this interpretation assuming that the N-terminal segment together with the minor loop constitute one domain and the major loop a second domain.

2) Formation or relaxation of the domain interactions in E2(K) and E1Na are paralleled in the phosphorylated conformations E2P and E1P and, we suggest, constitute an essential element of energy transduction. These interactions seem to occur within or near the active site, so explaining interference with cleavages of non-covalently or covalently bound phosphate or vanadate (Fig. 11). A change of microenvironment of bound phosphate from hydrophilic in high energy E1P to hydrophobic in low energy E2P has been proposed based on effects of organic solvents (46). The aqueous or less aqueous microenvironment of bound phosphate in E1P or E2P could be explained by the open or closed domain structure, respectively. A Mg2+ ion is tightly bound in E2P, and it can be proposed that tight co-ordination of the Mg2+ ion near TGES and VNDS and to the covalently bound phosphate induces the interactions between the minor and major loops which underlies the E1Pright-arrow E2P transition. In E2P, the bound phosphate (at CSDK and MVTGD) and the interacting sequences of the minor and major loops (TGES and VNDS) must lie fairly close to the membrane in order to explain proximity to HFIH near the entrance to M3. Accordingly, the high affinity ATP site in E1 should be organized such that the gamma -phosphate of ATP interacts near CSDK and MVTGD, while the purine binding residues (such as Lys501 and Gly502 (47, 48) and Lys480 (49)) are located toward the periphery of the major cytoplasmic loop.

3) Cation transport involves deocclusion of Na+ or K+ ions accompanying E1Pright-arrow E2P and E2(K)right-arrow E1, respectively, and requires opening or closing of "gates," which act as barriers to dissociation of cations. "Gates" could be formed by interacting residues of adjacent trans-membrane segments. In E1P(3Na) and E2(2K), "gates" are closed at both surfaces. In E2P, an extracellular "gate" is open and a cytoplasmic "gate" is closed, while in E1 the cytoplasmic "gate" is open and an extracellular "gate" is closed. We propose that formation or relaxation of interactions between cytoplasmic domains alters the twist or tilt or perhaps stretch of the relevant trans-membrane helices and so alters their mutual interactions, i.e. the "gates." Prime candidates as mobile trans-membrane segments are M4, M5, and M6, which contain residues involved in cation occlusion (7-9). Movement of M5, particularly Asn776, Ser775, Thr774, Thr771, and Tyr771 in the sequence YTLTSNIPEIT, seems to be important in the connection between the major loop and the membrane domain (50), as is Leu332 in the sequence 328PEGLL near the cytoplasmic boundary of M4 (51). There is evidence that M9 is a mobile segment, although the role of M7-M10 in cation occlusion is unclear (52). M1-M3 do not seem to play a role in cation occlusion, and indeed the lack of conformational sensitivity of the cleavages near M1 and M3 (HFIH) implies that M1 and M3 are static. The coupling mechanism described above is similar to a "scissors mechanism" proposed by Jørgensen et al. (53) with one significant difference. In Ref. 53, the handles of the scissors are represented by the sequences near Asp369 and Asp714 within the major cytoplasmic loop, which interact via the bound phosphate and Mg2+ ions. On our model, the handles represent the interacting minor and major loops as depicted in Ref. 13 and in Fig. 11. In both cases, the trans-membrane segments represent the blades of the scissors.

Conclusion-- Because the sequences involved in cytoplasmic domain interactions are highly conserved, the mechanism of energy coupling proposed here should apply to all P-type pumps. In this respect, it is remarkable that recent cryoelectron microscope images of Ca-ATPase indeed reveal a compact cytoplasmic domain in the E2 form but an open structure in the E1 form, with distinct lobes separated by a deep gorge (54).

    ACKNOWLEDGEMENTS

We are grateful to Dr. J. Kyte (University of California, San Diego, CA) for providing anti-KETYY and to Dr. R. Blostein (McGill University, Montreal, Canada) for providing rat axolemma and rat kidney microsomes.

    FOOTNOTES

* This work was supported by a grant from the Israel Science Foundation.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.

Dagger To whom correspondence should be addressed. Tel.: 972-8-934-2278; Fax: 972-8-934-4118; E-mail: bckarlis{at}weizmann.weizmann.ac.il.

2 If a fraction of added Fe2+ is bound to the membranes, ascorbate, etc., the true binding affinity of Fe2+ will be higher than the calculated affinity, but the relative difference in Rb+- versus Na+-containing media will remain. This possibility can only strengthen the argument against two Fe2+ sites.

3 D. Tal, J. Capasso, and S. J. D. Karlish, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: PVDF, polyvinylidene difluoride; PAGE, polyacrylamide gel electrophoresis; Tricine, N-tris(hydroxymethyl)methylglycine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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