Molecular Architecture of the Phosphorylation Region of the Yeast Plasma Membrane H+-ATPase*

Airat Valiakhmetov and David S. PerlinDagger

From the Public Health Research Institute, Newark, New Jersey 07103

Received for publication, August 30, 2002, and in revised form, November 12, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The molecular architecture of the yeast plasma membrane H+-ATPase phosphorylation region was explored by Fe2+-catalyzed cleavage. An ATP-Mg2+·Fe2+ complex was found to act as an affinity cleavage reagent in the presence of dithiothreitol/H2O2. Selective enzyme cleavage required bound adenine nucleotide, either ATP or ADP, in the presence of Mg2+. The fragment profile included a predominant N-terminal 61-kDa fragment, a minor 37-kDa fragment, and three prominent C-terminal fragments of 39, 36, and 30 kDa. The 61-kDa N-terminal and 39-kDa C-terminal fragments were predicted to originate from cleavage within the conserved MLT558GDAVG sequence. The 37-kDa fragment was consistent with cleavage within the S4/M4 sequence PVGLPA340V, while the 30-kDa and 36-kDa C-terminal fragments appeared to originate from cleavage in or around sequences D646TGIAVE and DMPGS595ELADF, respectively. The latter are spatially close to the highly conserved motif GD634GVND638APSL and conserved residues Thr558 and Lys615, which have been implicated in coordinating Mg2+ and ATP. Overall, these results demonstrate that Fe2+ associated with ATP and Mg2+ acts as an affinity cleavage agent of the H+-ATPase with backbone cleavage occurring in conserved regions known to coordinate metal-nucleotide complexes. This study provides support for a three-dimensional organization of the phosphorylation region of the yeast plasma membrane H+-ATPase that is consistent with, but not identical to, typical P-type enzymes.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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The fungal plasma membrane H+-ATPase is a typical P-type, class II, non-heavy metal-transporting enzyme. The catalytic cycle of the H+-ATPase, like all P-type membrane ATPases, comprises several transient phosphorylated forms that are tightly coupled to the binding or dissociation of transported ions at distant transport sites (1). The catalytic ATP hydrolysis domain is comprised of two interacting segments, a larger central segment of ~340 amino acid residues, extending from transmembrane segments M4-M5, containing the nucleotide-binding domain and site of phosphorylation (Asp378) and a smaller interacting domain of ~127 amino acid residues extending from M2-M3. Both domains contain large amounts of beta -strand and alternating beta -strand and alpha -helix regions as seen in the recent crystal structure of the related Ca2+-ATPase from sarcoplasmic reticulum (SERCA1a) (2). The catalytic region shows conservation with a large superfamily of hydrolases that are structurally typified by the L-2-haloacid dehalogenases (3, 4). Understanding the mechanism of these pumps will require atomic structures of several of the key conformational intermediates within the transport cycle. As a first step, it is important to confirm the molecular architecture of the nucleotide binding region in the related P-type pumps and dissect the subtle structural differences that contribute to their catalytic behavior.

The oxidative cleavage of proteins by oxygen radicals generated by tightly bound Fe2+(3+) or Cu2+ has been used to probe interacting protein structure elements in a number of nucleotide-binding proteins (5, 6). The process appears to involve cleavage of peptide bonds in close proximity to bound metals by the local formation of oxygen radicals, most likely OH radicals, by the Fenton reaction or a reactive metal-peroxyl derivative (7, 8). In the presence of Fe3+ or Cu2+, ascorbate or DTT1 promotes the generation of reactive oxygen species (·OH, O<UP><SUB>2</SUB><SUP>−</SUP></UP>, H2O2, and ferryl ion). Because several peptide bonds are cleaved from the same Fe2+ site, these cleavage positions must be in proximity in the native protein. For this reason, it has been useful in mapping the architecture of the active center of various metal-binding enzymes (5, 6). This approach is also highly sensitive to the conformational state of the protein and can be used to assess changes in interacting protein structure elements. Karlish and co-workers have used this approach of oxidative cleavage to probe the spatial organization of the renal Na+,K+-ATPase in defined E1 and E2 conformations (9-11). In this study, Fe2+ associated with ATP and Mg2+ has been used to probe the architecture of the phosphorylation region of the yeast plasma membrane H+-ATPase by analyzing peptide fragments generated by oxidative cleavage.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Strain Construction-- Recipient strain SH122 (HO ade6-1 trp5-1 leu2-1 lys1-1 ura3-1 pma1Delta ::LEU2/PMA1), derived from wild type strain Y55 (12), was used to construct a homozygous wild type strain containing PMA1 with an N-terminal histidine tag. All cells were grown until mid-log phase in YPD medium (1% yeast extract, 2% peptone, 2% dextrose, pH 5.7) at 30 °C.

Construction of the 10 Histidine-containing PMA1 Gene-- The PMA1 gene containing 10 consecutive histidine residues at the N terminus was constructed by a two-step site-directed mutagenesis approach using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). In the first mutagenesis step, six histidine residues were inserted between amino acids Ser5 and Ser6 in PMA1 contained on plasmid pGW201 using primer 5'-GACTGATACATCACATCATCATCATCATCATTCCTCTTCATCATCC. For convenience, silent mutations were engineered that provided a new BamH1 restriction endonuclease site. In the second step, an additional four histidines were added using primer 5'-GACTGATACATCACATCACCACCACCACCATCATCATCATCATTCC. The full construct was confirmed by DNA sequence analysis and introduced back into yeast by homologous recombination to create a homozygous mutant strain as previously described (12). All cells were grown in a 3-ml YPD culture for 18-20 h, and chromosomal DNA was isolated using the Wizard Genomic DNA Purification Kit (Promega). The PMA1 structural gene was amplified as a 3.5-kbp fragment by PCR using flanking primers (5'-GCTCCCCTCCATTAGTTTCG and 5'-GCGTGTTGTGAATTGTGC), and all mutations were verified by DNA sequence analysis of PCR-amplified products.

Standard Fe2+-catalyzed Cleavage Reaction-- A typical 50-µl reaction consisted of 50 µg of sucrose-gradient purified plasma membranes, 20 mM HEPES, pH 7, 20% (v/v) glycerol, 5 mM ATP, 5 mM MgSO4 and 20 µM FeSO4. The reaction was initiated by the simultaneous addition of 2 mM DTT and 2 mM H2O2 (final concentration), and it was stopped by the addition of a 5× SDS-containing sample preparation SDS-PAGE buffer. The samples were resolved on a NOVEX 10-20% gradient precast SDS-PAGE, and bands were visualized by Western blot analysis using anti-histidine tag (His6) antibody (Clontech) for the N terminus and anti-C-terminal peptide antibody (provided by Dr. Brian Monk, University of Otago, Dunedin, New Zealand). SDS-PAGE, blotting to polyvinylidene difluoride, and immunoblots Western blotting were performed in Xcell-II Blotting Module (NOVEX) in 10 mM CAPS, 10% (v/v) methanol, pH 11, at 480 mA for 1 h. Immunoblots were developed using an ECF Western blotting kit from Amersham with His6 monoclonal antibody at a dilution 1:25,000 as primary antibody and recorded using a STORM scanner (Molecular Dynamics).

Plasma Membrane Isolation and ATP Hydrolysis-- Plasma membranes were prepared from the His10 mutant strain by centrifugation on a sucrose step-gradient as previously described (13). ATPase assays were conducted in 96-well microplates, essentially as described by Monk et al. (14). The basic ATP hydrolysis assay medium consisted of 5 mM MgSO4, 5 mM ATP, 25 mM NH4Cl, and 10 mM MES-Tris, pH 6.5. The reaction was performed in 150 µl of volume with 1.0 µg of membrane protein at 30 °C for 20 min. The reaction was stopped by the addition of 100 µl of a combined Stop-Color Development reagent containing 1% SDS, 0.8% ascorbic acid, 100 mM ammonium molybdate, and 0.6 M H2SO4.

Purification of the Iron Cleavage Products on Ni-NTA Column-- Following Fe2+ cleavage, a 1-ml fraction (1 mg/ml total protein) was combined with 50 µl of 200 mM dodecyl maltoside and sonicated 10-30 s in a bath sonicator (80 watts; Laboratory Supply, Hicksville, NY). The 1-ml sample was transferred into a 15-ml falcon tube containing 5 ml of binding buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) and combined with 400 µl of aliquot of 50% Ni-NTA-agarose (Qiagen) slurry (200 µl of resin). The sample slurry was incubated for 2 h at 4 °C with gentle rotation (2 rpm). The samples were transferred to a Bio-Spin Disposable Chromatography Column (BioRad) and washed with 2 ml of binding buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) and 2 ml of washing buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0). The products were eluted four times with 200 µl of elution buffer (50 mM NaH2PO4, 300 mM NaCl, 1 mM dodecyl maltoside and 150 mM in place of 250 mM imidazole, pH 8.0). Each elution was collected, analyzed, and pooled.

Preparation of the Samples for MALDI-TOF Mass Spectrometry-- Samples were prepared according to Borchers et al. (15) with minor modifications. Pooled eluants from the Ni-NTA-agarose column were concentrated in a Speed-Vac concentrator (Savant Inc.) down to 40 µl. The samples were separated by gel electrophoresis on a 12% Laemmli gel, and peptides were stained with GELCODE Blue Stain (Pierce). Prominent bands at ~45 and 60 kDa were excised from the gel and placed into Eppendorf tubes (0.75 ml) containing a small needle hole at bottom. The tubes were placed in a 1.5-ml tube in a Microfuge and spun at top speed (~15,000 × g) for 5 min to force the gel slices through the small orifice. The disrupted gel slices were washed by centrifugation with 100 µl of distilled H2O. The gel was destained with five washes of 100 µl of acetonitrile/50 mM NH4HCO3, pH 8.6 or until coloration was removed. The gel slices were lyophilized in a Speed-Vac concentrator and rehydrated with 100 µl of 50 mM NH4HCO3. Samples were centrifuged for 5 min at 15,000 × g, and the supernatant was removed. An aliquot consisting of 50 µl of 25 mM NH4HCO3 and 0.02% SDS was added, and the mixture was incubated at 37 °C for ~18 h with shaking. Samples were centrifuged in a Microfuge at 15,000 × g for 5 min, and the supernatant was removed and concentrated down to 5-10 µl. SDS was added to a final concentration of 1% (w/v), and MALDI-TOF mass spectrometry was performed accordingly Breaux et al. (16).

Isolation of Secretory Vesicles-- Site-directed mutagenesis of residues Thr384, Lys615, Thr558, Asp634, and Asp638 were constructed in plasmid pRN202.1 (17) with the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Transformation of yeast strain SY4 (17) with plasmid DNA was performed with the Alkali-Cation Yeast Transformation Kit (Bio 101, Inc.), and secretory vesicles were isolated as described by Ambesi et al. (18).

Antibody Production-- Peptides corresponding to amino acid sequences 368LCSDKTGTLTKNKLS, 468PPFDPVSKKVTAVVES, 531PCMDPPRDDTAQTVS, and 628LVAMTGDGVNDAPSL were prepared by solid phase synthesis and were used for the preparation of rabbit antibodies (Sigma Genosys Co.). Enriched IgG fractions (Pierce) were validated against intact enzyme by Western blot analysis.

Other Procedures-- All mutations were identified by DNA sequence analysis at the New York University Medical Center Sequencing Facility. Protein concentrations were determined using the Coomassie Plus Protein Assay Reagent (Pierce). SDS-gel electrophoresis was performed using precast 10-20% minigels (Novex). Silver staining of protein gels was performed with GelCode SilverSNAP stain (Pierce).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Polyhistidine-marked Enzyme-- A PMA1-modified wild type strain containing a string of ten consecutive histidine residues between Ser5 and Ser6 at the N terminus of the H+-ATPase was used in this study. The polyhistidine-marked plasma membrane-bound enzyme behaved like wild type enzyme displaying a Km = 0.74 ± 0.1 mM and a Vmax = 4.45 ± 0.2 µmol Pi mg-1 min-1. The cells grew with a normal doubling time and were not sensitive to the presence of excess nickel in the growth medium. Likewise, ATP hydrolysis was not significantly altered by the binding of either an anti-His tag antibody or the presence of nickel at a 5-fold molar excess supporting a previous study showing that perturbations of the N terminus have little overall affect on the kinetic behavior of the yeast H+-ATPase (19). The polyhistidine enzyme was particularly useful because it allowed both N-terminal fragments to be easily detected by Western blot analysis with commercially available anti-histidine tagged antibody, and it enabled high abundance peptide fragments to be purified by nickel or Talon resin affinity chromatography.

Fe2+ Cleavage-- Incubation of the H+-ATPase with Fe2+(3+) in the presence of 2 mM H2O2, 2 mM DTT, 5 mM MgSO4 and 5 mM ATP resulted in a time-dependent cleavage of the enzyme as observed with antibodies directed against both N and C termini (Fig. 1). The N-terminal antibody showed enzyme cleavage and the appearance of several new bands ranging from 37 kDa to 61 kDa within the first time point at 5 min. (Fig. 1A). However, a predominant band at 61 kDa was observed as the major cleavage product. The C-terminal antibody also showed enzyme cleavage with three prominent C-terminal fragments estimated at 30, 36, and 39 kDa that were formed with the same kinetics as the appearance of a 61-kDa N-terminal fragment (Fig. 1B). In each case, the fragment profiles did not change, only the amount of product, suggesting that cleavage was occurring from a single locus and was not progressive. Silver-staining of the gels revealed that the 61-kDa peptide was the major cleavage product and that its abundance coincided with the decrease of 100-kDa intact H+-ATPase (Fig. 1C). The C-terminal fragments were more difficult to observe directly.


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Fig. 1.   Kinetics of Fe2+-catalyzed cleavage. Oxidative cleavage of the H+-ATPase was followed in a medium containing 20 mM HEPES, pH 7.0, 20% glycerol, plasma membranes (1 mg/ml), 20 µM FeSO4, 5 mM MgSO4, and 5 mM ATP. The reaction was initiated by the addition of 2 mM H2O2/DTT. At the times indicated, aliquots from the reaction were removed and diluted into SDS-gel electrophoresis sample buffer to stop the reaction. Cleavage products were separated by gel electrophoresis and analyzed by Western blot analysis with antibodies directed against the N terminus (anti-His tag) (A) or C terminus (peptide) (B). A parallel gel run under the same conditions was visualized directly with silver stain (C). The arrows indicate the position of prominent cleavage products.

Enzyme activity was also assessed under cleavage conditions. The addition of H2O2 (plus ascorbate) alone to the enzyme caused an immediate but variable decrease in ATP hydrolysis activity by 25-40%. However, in the presence of Fe2+, a further decrease in enzyme activity was observed that was proportional to the amount of total enzyme cleavage, as determined from the level of intact 100-kDa enzyme. Less than 10% activity was recovered after cleavage.

Specificity of Cleavage-- Specific enzyme cleavage resulting in characteristic fragment generation was dependent on ATP and Mg2+ and was optimal above the Km for ATP hydrolysis (>1 mM) as determined with N-terminal (Fig. 2A) and C-terminal antibodies (Fig. 2B). The high level of ATP required for significant cleavage appears to represents a slight modification in the affinity of the enzyme for nucleotide, which occurs following treatment with hydrogen peroxide. When Mg2+ was varied from 0 to 10 mM in the presence of 5 mM fixed ATP, the amount of characteristic 61-kDa cleavage product increased as the ratio of Mg2+ to ATP approached unity. Only trace cleavage was observed in the absence of added Mg2+ (Fig. 2C). At low Mg2+/ATP ratios, the 61-kDa fragment appeared as a doublet with the formation of a second, slightly higher 62-kDa fragment (Fig. 2C). This second fragment became much less prominent as the Mg2+/ATP ratio approached unity. At the higher ratios, minor fragments of 37 kDa and 72 kDa were also formed at detectable levels (Fig. 2A). It was further observed that two N-terminal fragments of about 65 and 67 kDa were observed under these conditions. However, these fragments were observed irregularly under comparable conditions and appeared to be related to normal breakdown of the enzyme. Two additional N-terminal fragments of 32 and 34 kDa were observed, but again their appearance was sporadic.


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Fig. 2.   Metal-nucleotide requirement for Fe2+-catalyzed cleavage. The efficiency of the Fe2+-mediated oxidative cleavage reaction was evaluated under standard reaction conditions in which the concentration of equimolar Mg2+ and ATP (A and B) was varied or the ratio of Mg2+ to ATP was varied (C). The reaction medium consisted of 20 mM HEPES, pH 7.0, 20% glycerol, plasma membranes (1 mg/ml), 20 µM FeSO4, and MgSO4, and ATP varied as indicated. Oxidative cleavage was allowed to proceed for 2 h, and samples were resolved by SDS gel electrophoresis and Western blot analysis with either N-terminal (anti-His tag) antibody (A) or C-terminal (peptide) antibody (B). In A and B, lane 1 (control): ATPase was mixed with SDS-sample buffer immediately; lane 2 (control): H2O2/DTT (2 mM) added alone; lane 3 (control): H2O2/DTT, Fe2+ added but not ATP and Mg2+. Lanes 4-9: H2O2/DTT, Fe2+, and increasing Mg-ATP concentrations. In C, MgSO4 was varied with a fixed amount of ATP (5 mM), and oxidative cleavage was evaluated with N-terminal (anti-His tag) antibody. Lane 1 (control): ATPase was mixed with SDS-sample buffer immediately; lane 2 (control): H2O2/DTT (2 mM) added alone; lanes 3-9: H2O2/DTT, Fe2+, and Mg2+ and ATP added, as indicated. The arrows indicate prominent bands.

The cleavage reaction was readily observed with both ATP and ADP but was not observed with GTP, CTP, TTP, AMP, or acetyl phosphate (ACP) (Fig. 3). The failure to detect cleavage with ACP is interesting because the H+-ATPase hydrolyzes this high energy substrate and forms a trypsin-detectable E2 conformation, but ACP cannot drive proton transport (20). The importance of bound nucleotide was further suggested by the finding that photoinactivation of the enzyme with 8-azido-ATP prior to cleavage with ATP eliminated selective cleavage (not shown).


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Fig. 3.   Substrate specificity for cleavage. Plasma membranes were incubated for 2 h with 20 mM HEPES, pH 7.0, 20% glycerol, 20 µM FeSO4, 5 mM MgSO4, 2 mM DTT/H2O2, and 5 mM ATP or other nucleotides as indicated, including acetyl phosphate (5 mM). The cleavage products were separated by gel electrophoresis and visualized with N-terminal (anti-His tag) antibody. Control samples (lanes 8-11) contained buffer, MgSO4, and DTT/H2O2 as indicated.

Fe2+-ATP complexes in the absence of added Mg2+ are weak substrates for ATP hydrolysis (<5%) by the H+-ATPase and were equally weak in inducing cleavage (Fig. 1), unlike their behavior with other P-type transport enzymes (9). An excess of free Mg2+ ions should displace Fe2+ from the ATP and suppress the cleavages from the ATP site. In fact, an excess of free Mg2+ over ATP reduced Fe2+-induced cleavage suggesting that an equilibrium complex of ATP-Mg2+·Fe2+ was most likely responsible for the specific cleavage (Fig. 2C).

In the Na+,K+-ATPase, free Fe2+ with H2O2 and ascorbate induces cleavage of the enzyme in the absence of nucleotide. The sequence 283HFIH near transmembrane segment 3 has been suggested to play a role in this binding (10). In the yeast H+-ATPase, free Fe2+ does not promote specific cleavage of the H+-ATPase. Increasing the amount of Fe2+ from 100-1000 µM results in nonspecific, generalized enzyme cleavage. The 283HFIH sequence in the Na+,K+-ATPase is 285HFTE at the equivalent position in the yeast H+-ATPase. Although, in the yeast enzyme, the region preceding this motif is disrupted by a seven-amino acid deletion. Genetic modification of PMA1 to 285HFIH at this position resulted in an active enzyme. However, the mutant enzyme was not selectively cleaved in the presence of free Fe2+ suggesting that the site, at best, has a loose affinity for Fe2+. It may be that the protein structure around the site in the yeast enzyme is sufficiently different to alter its metal binding affinity relative to the Na+,K+-ATPase. A second HFIH motif was engineered at amino acid position 683 in the large cytoplasmic loop. Again, the motif failed to bind free Fe2+ and induce selective cleavage. Both enzymes showed normal characteristic cleavage patterns in the presence of nucleotide.

The presence of nucleotide is absolutely required for cleavage, and the pattern of cleavage was unchanged in the presence of nucleotide with either vanadate (100 µM), NaF (1 mM), BeCl2 (10 µM) or NaF + BeCl2 (not shown), which all inhibited enzyme activity. In the case of BeCl2 + NaF, the kinetics for oxidative cleavage were much slower, but the pattern ultimately remained constant. There was no evidence for conformation-dependent changes in enzyme cleavage as has been observed in other systems (9, 10). These results suggest that bound nucleotide only is required to induce specific oxidative cleavage. It is likely that the E1 state is responsible for cleavage as its has the highest affinity for nucleotide.

Determining Fragment Identity-- The major N-terminal-marked fragments generated by Fe2+-catalyzed cleavage could be recovered by dodecyl maltoside extraction and isolation on a nickel affinity column. Unfortunately, N-terminal sequencing was of no value because cleavage occurred at the C-terminal end of each peptide and the polyhistidine tag was at the N terminus. Detergent extraction of the cleaved fragments and recovery by nickel column chromatography yielded fragments that were analyzed by MALDI-TOF mass spectrometry. The fragments were easily observed by SDS-PAGE but were often poorly detected by mass spectrometry. Nonetheless, in several experiments, fragment sizes of 61,479, 62,162, 62,424, and 37,488 could be identified, which corresponded to cleavage in the regions around amino acids TGD570AVGIAKE577 and PVGLPA340.

To further confirm the regions covered by the various fragments, peptide antibodies were prepared to critical regions of the enzyme as follows: 368LCSDKTGTLTKNKLS (P368), 468PPFDPVSKKVTAVVES (P468), 531PCMDPPRDDTAQTVS (P531), and 628LVAMTGDGVNDAPSL (P628). The antibodies were used to determine whether the linear sequence epitopes were present in the different fragments by Western blot analysis. Table I shows results from Western blot analysis of fragments. Antibodies P368, P468, and P531 reacted with the 57- and 61-kDa fragments, while the 30-, 36-, and 39-kDa fragments interacted with P628. The 39-kDa fragment was recognized by the P531 and P628 antibodies.

                              
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Table I
Interaction of peptide antibodies with major cleavage products
Western blot analysis was used to assess interactions between peptide antibodies 368LCSDKTGTLTKNKLS (P368), 468PPFDPVSKKVTAVVES (P468), 531PCMDPPRDDTAQTVS (P531), and 628LVAMTGDGVNDAPSL (P628) and major cleavage products. Strong positive responses are demoted by ++.

Modification of Coordinating Residues-- An important outcome of the high resolution model for the SR Ca2+-ATPase is that the nucleotide binding domain approximates the fold observed for the L-2-haloacid dehalogenase enzymes, which has been used as a structural template for P-type enzymes (4, 21). A number of key residues including Thr384, Lys615, Thr558, and Asp634 and evidence from other P-type enzymes support the key role of these amino acids in catalytic function, but such evidence was not available from the yeast H+-ATPase. To address the critical role of these amino acids, we introduced both conservative and non-conservative mutations at these positions and expressed the mutants in a secretory vesicle system. Substitution of Lys615 with Ala, Leu, Gln, or Arg resulted in highly defective enzymes that assembled at <15%. Substitution of Thr384 and Thr558 with Ser resulted in mutant enzymes with 44 and 25% activity, respectively, while substitution of Asp634 and Asp638 with Asn resulted in 19 and 22% residual activity, respectively. Even when corrected for level of assembly (<15-50%), the activity of each mutant was significantly altered by each of the mutations. The data support the notion that these residues play an important role in maintaining catalytic site integrity, which is necessary for catalysis and assembly.

    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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REFERENCES

Fe2+-catalyzed cleavage has been used as a valuable tool to explore local protein structure interactions associated with binding of free metal and metal-nucleotide complexes in animal P-type ATPases (5, 9, 11, 22, 23). In this study, metal associated with nucleotide was used to explore the molecular architecture of the phosphorylation domain of the yeast plasma membrane H+-ATPase. Free metal produced nonspecific enzyme degradation, while selective cleavage occurred only under conditions that promoted ATP binding (Fig. 2). Only ATP and ADP in the presence of Mg2+ and Fe2+ produced selective cleavage events. An E1 state was sufficient for cleavage as trapping the enzyme in other conformational states with vanadate or with transition state mimics such as fluoroaluminate or beryllofluoride had no influence on the cleavage pattern. Blocking the adenine binding pocket by pretreatment of the H+-ATPase with 8-azido-ATP abolished cleavage with ATP, while neither inorganic phosphate nor acetyl phosphate, which is readily hydrolyzed by the H+-ATPase (20), promoted cleavage in the presence of Fe2+. Unlike other P-type enzymes, Fe2+ substitutes poorly for Mg2+ in the hydrolysis reaction (1-2% of maximal activity) presumably because nucleotide binding, which is normally weak (Kd ~ mM range) under optimal conditions (equimolar Mg2+ and ATP), is diminished in the absence of Mg2+. Trace amounts of iron-induced cleavage were observed in the absence of Mg2+, even with equimolar ATP and Fe2+ in the mM range, but maximal cleavage was obtained only under optimal nucleotide binding conditions. Excess Mg2+ reduced the cleavage reaction as the molar amount of Mg2+ was increased over that of ATP at a fixed Fe2+ concentration. It is important to note that the cleavage pattern did not change with time, rather the intensity of each fragment increased (Fig. 1) suggesting that the cleavage reaction occurred at a fixed locus. In addition, since large stable fragments were generated, it is likely that the metal-nucleotide complex is lost following the initial cleavage events. Thus, the fragments produced effectively defined a fixed conformational state.

The architecture of the nucleotide binding and phosphorylation domains of P-type enzymes has been illuminated in recent years by the recognition of an apparent similarity with the haloacid dehalogenase and related enzymes (4, 21, 24) and more significantly by the emergence of a 2.6-Å high resolution structure of the Ca2+-ATPase (2). Critical residues responsible for coordinating metal-nucleotide complexes have been identified in conserved sequence motifs, which for yeast PMA1 include CSD378KTGTLT, containing the site of phosphorylation (Asp378), RVXMLT558GD, PQXK615XXVVE, and TGD634GVND638APXL. These sequence stretches all lie within the large central cytoplasmic domain bordered by M4 and M5. The spatial organization of these critical residues for ATP binding and hydrolysis are shown in Fig. 4, which represents a superposition model of the yeast H+-ATPase on a SERCA1a template (25). The phosphorylation site (Asp378) is >25 Å away from the bound nucleotide, and domain closure within this region is presumed to occur to facilitate phosphate transfer (2). Additional domain movements occur during catalysis that bring other conserved sequences into close proximity with this region, such as TGE233S on the cytoplasmic loop between M2 and M3 (9).


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Fig. 4.   Molecular model of H+-ATPase catalytic region. The primary amino acid sequence of yeast PMA1p from Ala326-Arg751 was overlaid on the molecular structure coordinates for the SERCA1a as previously described (25). The view is from the center of the enzyme at the cytoplasmic face toward the bilayer. The backbone trace shows identical residues (yellow), conserved residues (orange), non-conserved residues (black), and absent residues (white), which are not present in the yeast H+-ATPase. Notable residues include the site of phosphorylation, Asp378, and residues Thr558, Lys615, Asp634, and Asp638, which help coordinate substrate and Mg2+ binding and phosphoryl transfer (21). Amino acid sequences implicated as cleavage sites from fragment analysis are indicated.

Since Fe2+ cleavage is dependent on bound nucleotide, it is likely that the cleavage sites should help define protein structure in close proximity to the nucleotide, which should better define similarities and differences between the yeast H+-ATPase and related P-type enzymes. Antibody directed to the N terminus of PMA1 revealed a prominent fragment at ~61 kDa and a minor fragment at 37 kDa. A C-terminal-directed antibody identified three prominent fragments of 30, 36, and 39 kDa. The N-terminal fragments, which were marked with a polyhistidine tag, were purified by nickel chromatography, and analysis of the fragments by MALDI-TOF mass spectroscopy yielded two prominent fragment masses of 61,479-62,351 Da and 37,488 Da. Given these apparent masses, it can be assumed that cleavage resulting in the 61-kDa fragment occurred within the region consisting of amino acids MLT558GDAVG, which includes the highly conserved Thr558. The minor 37-kDa fragment represented cleavage within PVGLPA340V, which is near the interface region between membrane-spanning helix 4 (M4) and stalk 4 (S4). The prominent C-terminal fragments were more difficult to isolate. Their sizes and identity could only be inferred from their migration behavior by SDS-gel electrophoresis (Figs. 1 and 2) and peptide antibody evaluation (Table I). The 30-kDa fragment would correspond to cleavage around D646TGIAVE, which is near to the highly conserved motif GD634GVND638APSL. The 36-kDa fragment would correspond to the region DMPGS595ELADF, which lies between the regions containing conserved residues Thr558 and Lys615. Interestingly, 22 residues of SERCA1a in this region are absent in PMA1, which would tend to bring these two highly conserved residues closer together in real space. Finally, the 39-kDa fragment would correspond to cleavage within LT558GDAVGIAKET, which contains the highly conserved Thr558 and is consistent with cleavage producing the 61-kDa N-terminal fragment.

It is noteworthy that prominent cleavage occurred in or around conserved regions GXRVXKLT558GD, D591MPGS, and GD634GVND638APSLKK suggesting that these regions were in most direct contact to the bound Fe2+-nucleotide complex. Fig. 4 shows the close proximity of this region to the site of phosphorylation (ASP378). Residues Thr558, Asp634, and Asp638 in these sequences help coordinate the metal-nucleotide complex, as is apparent in the phosphorylation domain of haloacid dehalogenases (2, 4, 21) In this model, substrate binding is mediated by Thr558 and a Mg2+ ion, which presumably shields the negatively charged phosphate group. Mg2+ is believed to be coordinated by oxygen atoms from Asp378 and Asp634 (21). In the Na+,K+-ATPase, the conserved sequence TGD710GVND contributes to coordination of Mg2+ during transfer of gamma -phosphate to Asp369 in the E1 form of the enzyme. In contrast, the transition from E1P to E2P in coupling with Na+ is accompanied by a shift of Mg2+ binding away from Asp710 and Asn713 (26). Lys615 is expected to help coordinate the carboxylate group of Asp378 in the enzyme-substrate complex. It is likely that in the presence of nucleotide, Fe2+ and Mg2+ are complexed by the key residues described in this local structure. As further validation, conserved amino acids Thr384, Asp634, Asp638, Thr558, and Lys615 in PMA1 were modified by site-directed mutagenesis. In all cases, conservative and non-conservative substitutions resulted in reduced activity even when enzymes were poorly assembled, confirming the importance of these residues for enzymatic function in H+-ATPase. Overall, these data suggest that the phosphorylation domain of the yeast H+-ATPase conforms to typical P-type spatial architecture.

Despite the gross spatial similarity of coordinating residues, subtle differences in catalytic site architecture are expected given the kinetic differences between the H+-ATPase and related P-type family members. Some of these differences are apparent in the superposition model (Fig. 4) where the backbone trace of the SERCA1a pump is used to display conserved, non-conserved, and absent residues. The model serves to illustrate that while overall features are conserved, important differences are apparent that distinguish the two enzymes and most likely explain their differential kinetic properties. While considerable structural homology exists between the various enzymes, there are important differences between the N and P domains. In particular, the large amount of diversity in the nucleotide binding domain of the enzyme, including large stretches of absent sequence in PMA1 is striking. The affinity of the H+-ATPase for ATP is considerably weaker than its animal cell counterparts. When compared with the SERCA1a enzyme, ~24% of the structure from Ala326 near the interface of M4 to Arg751 near M5 (more than 100 residues) is absent in the yeast H+-ATPase. These large deletions are expected to contribute to the kinetic diversity observed between H+-ATPase and other P-type pumps (26).

The adenosine binding pocket in the N-domain is reasonably well defined. Derivatization of Lys492 or Lys515 (SERCA1a) interferes specifically with binding and delivery of ATP substrate to the catalytic site but does not necessarily disrupt the catalytic mechanism. Yet binding of a reagent like 4-diisothioryano-2,2 disulfonic acid stilbene (DIDS) to these residues effectively closes the nucleotide pocket and prevents proper interaction of the N and P domains (27). Both Lys492 and Lys515 are conserved in the yeast H+-ATPase as is Arg511, which is equivalent to Arg544 (Na+,K+-ATPase) or Arg560 (SERCA1a). These residues appear to play a critical role in ATP binding in the Na+,K+-ATPase (28) and Ca2+-ATPase (27). In the Na+,K+-ATPase, reduced affinity for ATP was observed with mutation of residue Arg544, as well as nearby residues Asp555, Glu556, Asp565, and Asp567 (29). It is noteworthy that the region Leu551-Glu568, which is upstream from the cluster of residues important for nucleoside binding and which contains most of these acidic residues, is absent in the yeast PMA1 enzyme.

It is also important to note that there are some significant differences between our findings on the yeast H+-ATPase and those of Karlish and colleagues on the Na+,K+-ATPase (5, 9, 11, 22, 23). Firstly, there is no evidence for a metal binding site in the absence of nucleotide. In the Na+,K+-ATPase, it was suggested that Fe2+ binding occurs at a site, HFIH, in stalk segment 3 just above M3. However, we created the equivalent site in the yeast PMA1 (286HFTE right-arrow HFIH) but failed to detect any specific cleavage at this or any other site even at elevated Fe2+ levels (>500 µM). More significantly, oxidative cleavage was observed in the conserved motif TGE214SE in the Na+,K+-ATPase in the E2 state providing strong evidence for interacting domains (9). Similar evidence for interacting domains was not obtained in our study. The simplest interpretation is that Fe2+ associated with nucleotide was not in close enough proximity to the interacting loop bound by M2 and M3 during catalysis even when the enzyme was inhibited by vanadate, BeF3, or AlF3.

Finally, a minor N-terminal cleavage product of 37 kDa was readily observed (Figs. 1 and 2) and could be purified by polyhistidine affinity chromatography. Surprisingly, cleavage is predicted to occur within PVGLPA340V, which is near the interface region between membrane-spanning helix 4 (M4) and stalk 4 (S4). This result suggests that this region during catalysis may move closer to the site of phosphorylation than would be anticipated, perhaps further indicating that M4 also undergoes a significant displacement.

    ACKNOWLEDGEMENT

We are grateful to Steven J. D. Karlish for helpful discussions and advice.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM 38225 (to D. S. P.).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: Public Health Research Institute, 225 Warren St., Newark, NJ 07103. Tel.: 973-854-3200; Fax: 973-854-3101; E-mail: perlin@phri.org.

Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.M208927200

    ABBREVIATIONS

The abbreviations used are: DTT, dithiothreitol; CAPS, 3-(cyclohexylamino)propanesulfonic acid; MES, 4-morpholineethanesulfonic acid; Ni-NTA, nickel-nitrilotriacetic acid; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; ACP, acetyl phosphate; SERCA1a, calcium ATPase of skeletal muscle sarcoplasmic reticulum.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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