©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Changing the Ion Binding Specificity of the Escherichia coli H-transporting ATP Synthase by Directed Mutagenesis of Subunit c(*)

(Received for publication, October 7, 1994)

Ying Zhang (§) Robert H. Fillingame (¶)

From the Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Most F(1)F(0) type ATP synthases, including that in Escherichia coli, use H as the coupling ion for ATP synthesis. However, the structurally related F(1)F(0) ATP synthase in Propionigenium modestum uses Na instead. The binding site for Na resides in the F(0) sector of the P. modestum enzyme. We postulated that Na might interact with subunit c of F(0). Subunit c of P. modestum and E. coli are reasonably homologous (19% identity) but show striking variations around the H-translocating, dicyclohexylcarbodiimide-reactive carboxyl (Asp in E. coli). Several hydrophobic residues around Asp were replaced with polar residues according to the P. modestum sequence in the hope that the polar replacements might provide liganding groups for Na. One mutant from 31 different mutation combinations did generate an active enzyme that binds Li, the combination being V60A, D61E, A62S, and I63T. Li binding was detected by Li inhibition of ATP-driven H transport, Li inhibition of F(1)F(0)-ATPase activity, and Li inhibition of F(0)-mediated H transport. The Li effects were observed with membrane vesicles prepared from a DeltanhaA, DeltanhaB mutant background which lacks Na/H antiporters, and with purified, reconstituted preparations of F(0) prepared from this background strain. Li inhibition was observed at pH 8.5 but not at pH 7.0. H thus appears to compete with Li for the binding site. Li binding was abolished by replacement of Glu by Asp or Ser by Ala. The side chains at Ala and Thr may act in a supporting structural role by providing a more flexible conformation for the Li binding cavity. Thr does not appear to provide a liganding group since H transport in two other mutants, with Gly or Ala in place of Thr, was also inhibited by Li. We suggest that a X-Glu-Ser-Y or X-Glu-Thr-Y sequence may provide a general structural motif for monovalent cation binding, and that the flexibility provided by residues X and Y will prove crucial to this structure.


INTRODUCTION

The F(1)F(0)-type H-transporting ATP synthases catalyze the synthesis of ATP during oxidative and photophosphorylation in a variety of organisms, usually using a transmembrane electrochemical H gradient as the driving force for ATP synthesis (Senior, 1988). These enzymes are composed of two sectors termed F(1) and F(0). The F(1) sector is easily released from the membrane and, when isolated, functions as an ATPase. The F(0) sector traverses the membrane and functions in H transport. The complete coupled enzyme functions as a reversible, H-transporting ATP synthase. A typical F(1)F(0) complex is found in Escherichia coli, where F(1) is composed of five types of subunits in an alpha(3)beta(3) stoichiometry, and F(0) is composed of three types of subunits in an a(1)b(2)c stoichiometry (Foster and Fillingame, 1982).

In the bacterium Propionigenium modestum, a profound variant of the F(1)F(0)-type ATP synthase is found which uses Na instead of H as the coupling ion for ATP synthesis (Laubinger and Dimroth, 1987, 1988). The size and composition of P. modestum subunits are very similar to that in E. coli. The enzymes also share many sequence homologies, i.e. the sequence identity between the most conserved subunit beta is 69% and the identities between the F(0) subunits a, b, and c are 25, 29, 19%, respectively (Ludwig et al., 1990; Esser et al., 1990; Kaim, et al., 1992). The F(1)F(0) complex of P. modestum, but not F(1) alone, is activated by Na or Li, which suggests that the cation binding site is in the F(0) moiety (Laubinger and Dimroth, 1987). This conclusion is further substantiated by the properties of a hybrid enzyme composed of the F(1) from E. coli and the F(0) from P. modestum, formed both in vitro (Laubinger et al., 1990) and in vivo (Kaim et al., 1992; Kaim and Dimroth, 1993). The hybrid enzyme shows Na activation of ATPase activity and functions as a Na pump. At Na concentrations < 1 mM, H and Na compete for the binding and transport by the P. modestum F(1)F(0) (Laubinger and Dimroth, 1989). In addition, both Na and Li inhibit passive H translocation by P. modestum F(0) (Kluge and Dimroth, 1992). Dicyclohexylcarbodiimide (DCCD) (^1)reacts specifically with the Asp of E. coli subunit c, or a Glu at the equivalent position of other F(1)F(0) complexes, and thereby blocks H translocation through F(0) and coupled ATPase activity (Hoppe and Sebald, 1984; Fillingame 1990). Subunit c of P. modestum F(0) also reacts with DCCD, presumably by covalently binding at the Glu residue (Laubinger and Dimroth, 1988). The reaction with DCCD is blocked when Na or Li is bound to the P. modestum F(0) (Kluge and Dimroth, 1993a, 1993b, 1994).

Subunit c is thought to fold in the membrane in a hairpin-like structure of two alpha-helices. Asp of E. coli subunit c is postulated to protonate and deprotonate during each cycle of F(0)-mediated H transport (Fillingame, 1990), and Glu of P. modestum subunit c is now thought to be a key component of the Na-binding site (Kluge and Dimroth, 1993a, 1993b, 1994). Comparison of the amino acid sequences of E. coli and P. modestum subunit c reveals striking variations around the DCCD-reactive carboxyl group (Fig. 1). Several hydrophobic residues in E. coli subunit c, which are also hydrophobic in other species (Hoppe and Sebald, 1984), are found as polar residues in P. modestum subunit c. These polar residues could serve as liganding groups to bind Na. The E. coli residues with polar replacements include Ile, Met, Ala, and Ile. The E. coli Pro, which is conserved in most other bacteria, is replaced by Gly and the nearby invariant Val by Ala. Genetic studies of E. coli subunit c indicate that a Pro is required in either of the two membrane-spanning helices (Fimmel et al., 1983; Fraga, 1990). In the P. modestum subunit, Pro is found at the equivalent of E. coli position 24 and Gln at the equivalent of position 28; this pattern is also seen in the chloroplast subunit c.


Figure 1: Helical hairpin model for folding of subunits c from E. coli and P. modestum. The rectangles encase the proposed transmembrane helices. Identical residues in the protein of both species are indicated by the closed spheres. Residue interchanges of Ala for Gly, or between Leu, Ile, and Val are indicated by hatched spheres. The polar loop region of the protein, including Arg in E. coli subunit c, faces the cytoplasmic side of the inner membrane. Single-letter abbreviations indicate residues surrounding the conserved Asp of E. coli subunit c which were replaced by equivalent residues from the P. modestum sequence.



In this study, we made a series of P. modestum-like substitutions in E. coli subunit c in the hope of generating an E. coli enzyme that would bind Na. From a combination of mutations, we ended up generating an active enzyme that is inhibited on binding Li.


EXPERIMENTAL PROCEDURES

Oligonucleotide-directed Mutagenesis and Plasmid Construction

Oligonucleotide-directed mutagenesis was initially carried out on phage F3, a M13mp19 derivative containing a PstI(1561)-PvuII(2376) unc DNA, (^2)by the method of Taylor et al.(1985) using a mutagenesis kit from Amersham Corp. A 40-base antisense oligonucleotide (5`-TACAGCGATCATCGSGGTAGAWTCCGCCAGACCCAT) (S = C, G; W = A, T; underlined bases are those mutated) was used to generate the E239-242 mutations (see Table 1), and another 40-base antisense oligonucleotide (5`-CCCGAGGATGCCTTGACCGATCGCAGGAC CGATTGC) was used to generate the E238 mutation. The PstI (1561)-HpaI(2162) fragment of mutated unc DNA was then cloned into equivalent sites of plasmid pDF163 (Fraga et al., 1994), which is a pBR322 derivative carrying the uncBEFH (bases 870-3216) genes. The other mutations were introduced by the polymerase chain reaction as described by Herlitze et al.(1990) with several 21-base oligonucleotides, using either wild type pDF163 or its mutant plasmid derivatives as DNA templates. The mutated polymerase chain reaction fragments were then digested with PstI and HpaI and ligated into the equivalent sites of pDF163.



Plasmid pYZ209, containing the eight structural genes of unc operon, i.e. unc DNA from HindIII (870) to BglII (10,172), was constructed by ligation of the SphI fragment of DNA (bases 3216-10,172 coding the uncAGDC genes) from plasmid pAP55 (Brusilow et al., 1983) into the unique SphI site of plasmid pYZ126, which is the uncE239 equivalent of plasmid pDF163. Plasmid pMO142 (Zhang et al., 1994), the wild type equivalent of plasmid pYZ209, is the control plasmid used in this study.

Complementation of uncE Mutations

Strain DF514 (uncE1003 (L4amber), F`lacIq) (Fraga and Fillingame, 1991) or strain AN936 (uncE429 (G23D); Downie et al., 1979) was transformed with pDF163 and its mutant derivatives. Transformant colonies were transferred with toothpicks as patches to minimal medium agar plates containing 22 mM succinate as carbon source and incubated at 37 °C with scoring for growth after 1-5 days.

Incorporation of Mutated uncE Genes into Chromosome

DNA from plasmid pDF163, and its succinate-plus mutant derivatives, was crossed into the chromosome of the uncE deletion strain LW180 (DeltauncE334) by a double recombination as described (Miller et al., 1989). The recombinant chromosomal uncE gene was transferred by P1 transduction with Ilv into strain MJM63 (AN346, DeltauncE334, ilv:Tn10) (Miller et al., 1989) or strain YZ323 (DeltauncB-C, ilv:Tn10, melBlid, DeltanhaA, DeltanhaB, DeltalacZY, thr1). Strain YZ323 was constructed by P1 cotransduction of DeltauncB-C and ilv:Tn10 from strain DK8 (Klionsky et al., 1984) into strain EP432 (melBlid, DeltanhaA, DeltanhaB, DeltalacZY, thr1) (Pinner et al., 1993). Strain EP432 was a generous gift from Drs. E. Padan and S. Schuldiner (The Hebrew University of Jerusalem, Israel). The chromosomal DNA of these transductants was amplified by the polymerase chain reaction and the DNA sequenced with an fmol Sequencing System (Promega Corp., Madison, WI) to confirm transfer of the uncE mutations.

Membrane Preparations, Assays, and Buffers

Cells for biochemical characterization were grown on M63 minimal medium supplemented with 1% D-glucose, 1 g/liter tryptone, 0.5 g/liter yeast extract, 87 mM KCl, 2 µg/ml thiamine, and 0.4 mMDL-threonine and harvested in the late exponential phase of growth. When required, antibiotics were added to final concentration of 100 µg/ml for ampicillin, 50 µg/ml for kanamycin, and 30 µg/ml for chloramphenicol. Membranes were prepared in TMDG buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl(2), 1 mM dithiothreitol, and 10% glycerol) by rupture of cells in a French press (Mosher et al., 1985).

Standard HMK buffer contains 10 mM HEPES-KOH, pH 7.5, 5 mM MgCl(2), and 300 mM KCl. Modified HMK buffers were prepared in which part of KCl was replaced by LiCl or NaCl at the concentrations specified, and the pH adjusted to 7.0, 7.8, or 8.5. These buffers were used in the ATP-driven ACMA quenching and ATPase assays. Fluorescence measurements were made as described in Zhang and Fillingame(1994). ATPase activity was assayed at 25 °C with 1 mM Tris-ATP [-32] adjusted to pH 7.5 with KOH. Protein was determined with a modified Lowry assay as described (Fillingame, 1975).

Reconstitution of Proteoliposomes with Isolated F(0)

Diploid strain YZ439 (pYZ209/uncE239, DeltanhaA, DeltanhaB) and the wild type control strain YZ418 (pMO142/uncE, DeltanhaA, DeltanhaB) were used as sources of F(0). F(0) was isolated according to the method of Schneider and Altendorf(1984) and stored at -80 °C in a buffer containing 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, and 1% sodium-cholate after freezing in liquid nitrogen. Phosphatidylcholine (Sigma, Type II-S), acetone washed according to Sone et al.(1977), was suspended at 40 mg/ml in a buffer containing 15 mM Tricine-KOH, pH 8.0, 0.2 mM EDTA, 7.5 mM dithiothreitol, 0.8% sodium deoxylcholate, 1.6% sodium cholate, and mixed with equal volume of purified F(0) in a protein to lipid ratio of 1:80 or 1:640. The mixture was dialyzed overnight at 4 °C against 1000 volumes of buffer containing 5 mM bis-tris-propane, pH 8.4, 2.5 mM MgCl(2), 0.2 mM EDTA, 0.2 mM dithiothreitol. The dialyzed sample were loaded with KCl by mixing with equal volume of a buffer containing 0.4 M KCl, 5 mM bis-tris-propane, pH 8.4, and 1 mM MgCl(2). The mixture was sonicated under a stream of argon at 0 °C, frozen in liquid nitrogen for 10 min, and thawed at room temperature. The F(0)-containing proteoliposomes obtained were then concentrated by centrifugation at 100,000 times g for 45 min, resuspended in a buffer containing 0.2 M KCl, 5 mM bis-tris-propane, pH 8.4, and 1 mM MgCl(2), and passed through the sonication-freeze-thaw procedure as described above. H uptake by the K-loaded proteoliposomes was measured by the quenching of ACMA fluorescence in a buffer containing 5 mM bis-tris-propane, pH 8.4, 0.2 M choline-Cl, and 1 mM MgCl(2) after adding valinomycin to 10 nM to induce K diffusion.


RESULTS

Substitutions and Complementation Test for Function

P. modestum subunit c contains a phylogenetically conserved Glu at the position equivalent to Asp of E. coli subunit c. Miller et al. (1990) have shown that replacement of Asp with the Glu greatly reduces the proton pumping function of the E. coli enzyme. We replaced amino acids at several positions in the vicinity of residue 61 with the residues found at equivalent positions in P. modestum subunit c, with the intention that these changes might both optimize function of the D61E mutant and alter the ion specificity.

A total of 33 mutants were generated by oligonucleotide-directed mutagenesis. These mutants fall into several groups according to their ability to complement the chromosomal L4amber or G23D subunit c mutations as scored by growth on a succinate carbon source (Table 1). Replacement of Ile with a polar residue Thr improves the function of D61E mutant (see E275, group 1) and also compensates for an otherwise deleterious effect of the A62S mutation (compare E276 and E274). An additional change of V60A (see group 2) greatly enhances the effect of I63T substitution and restores growth via oxidative phosphorylation to a wild type level (see E272 and E239). The V60A, A62S, I63T (E240) triple mutant, with Asp at position 61, grew less well. These results indicate that the longer side chain of the Glu residue can be accommodated by a more polar microenvironment and/or more flexible structure. Two other mutants containing substitutions of V60A, D61E, A62S, I63G, or I63A (E277 and E278) were also generated. Both displayed hearty growth on a succinate carbon source which suggests a need for a flexible rather than polar residue at position 63.

Single residue replacements of Ile or Met with Gln generate an active enzyme (see E249 and E244 under groups 3 and 4). However, these mutations abolished growth when they were combined with any of the other substitutions (E245-248 in group 3 and E260-263 in group 4). Pro is essential for function in the absence of compensatory substitutions of Pro at position 20 of helix-1, i.e. a second mutation of A20P in helix-1 can partially suppress mutation of P64L or P64A in helix-2 (Fimmel et al., 1983; Fraga, 1990). However, the possibly similar exchange of Pro with Ala of helix-1, as is found in the aligned sequence of the P.modestum subunit c, gave rise to nonfunctional proteins (see group 5). On the other hand, exchange of Pro of helix-2 with Ala of helix-1 did restore some function to the mutant enzyme containing a P.modestum-like pocket, i.e. A60E61S62T63 (^3)(compare E266 in group 6 to E264 in group 2).

Growth Properties of Chromosomal Strains

Nine of the mutations showing positive complementation were integrated into the chromosome as a prerequisite to their biochemical characterization. This was done to avoid potential problems with F(0) assembly which can result from expression of all or part of the unc operon from plasmids (Fraga et al., 1994). These chromosomal mutations were then transferred by P1 transduction into the strains with either an AN346 background or a Na/H antiporter-deficient (DeltanhaA, DeltanhaB) background. The chromosomal mutations supported growth similarly in either background (Table 2). The set of strains in the DeltanhaA, DeltanhaB background was chosen for further biochemical characterization to avoid possible artifacts from the Na/H antiporters.



ATPase-coupled Proton Translocation

ATPase-coupled proton translocation into membrane vesicles was measured by the quenching of ACMA fluorescence. In the experiments shown in Fig. 2, at pH 8.5, the extent of quenching response decreases in the order: wild type > A60E61A62T63 approx V60E61A62T63 approx A60E61A62I63 approx V60E61S62T63 approx A60E61S62T63 > V60E61A62I63 > A60D61S62T63. The order corresponds generally with the relative size of single colonies growing on a succinate carbon source. No quenching response was observed for P20A60E61S62T63G64 membranes (not shown). The quenching response of the A60E61S62T63 membranes was dramatically inhibited by Li, while quenching by wild type and the other mutant membranes was not affected by Li. ATP-driven ACMA quenching by A60E61S62T63 membranes was inhibited 80% by 50 mM LiCl, whereas 50 mM NaCl had no effect. It should be noted that two mutant membranes, V60E61A62I63 and A60D61S62T63, which showed a smaller ATP-driven ACMA quenching response than A60E61S62T63 membranes, were unaffected by 50 mM Li. The inhibition by Li of ATP-dependent proton translocation with the A60E61S62T63 membranes therefore can not be ascribed to the relatively low activity. The results do suggest that a Li-specific binding site may have been formed in the A60E61S62T63 mutant F(0) and that Li binding is manifested by inhibition of ATP-driven H translocation.


Figure 2: ATP-driven quenching of ACMA fluorescence by wild type and mutant membranes at pH 8.5. Membranes were diluted to 0.25 mg/ml in HMK buffer, pH 8.5, prepared with or without 50 mM LiCl or NaCl replacing a portion of the KCl. ACMA was added to 0.3 µg/ml and the fluorescence quenching initiated by addition of Tris-ATP to 0.9 mM. SF6847 was added to 0.3 µM.



All four substitutions in the A60E61S62T63 mutant may be necessary for the Li binding. The quenching response of membranes from the V60E61S62T63, A60D61S62T63 and A60E61A62T63 mutants was not inhibited by Li. To address the question of whether the change of I63T was absolutely required for the Li effect, two other mutants were constructed containing mutations of A60E61S62G63 and A60E61S62A63. ATP-driven ACMA quenching by these two membranes was also inhibited by LiCl (as shown in Fig. 3, B and C), but not by Na. Thr is thus not required for Li binding. Rather, a smaller and perhaps more flexible residue may be required in place of the Ile residue.


Figure 3: pH dependence of Li inhibition of ATP-driven quenching of ACMA fluorescence. Membranes were diluted to 0.25 mg/ml in HMK buffer at the pH and LiCl or NaCl concentration specified. The other conditions are as described in Fig. 2.



The effect of Li ions on the quenching response of A60E61S62T63, A60E61S62G63 and A60E61S62A63 membranes was markedly influenced by the pH of the assay buffer, as shown in Fig. 3. The Li inhibition of ATP-driven fluorescence quenching decreased as pH decreased. At pH 7.8, 50 mM Li inhibited quenching of A60E61S62T63 membranes by only 35% in contrast to the 80% inhibition at pH 8.5. At pH 7.0, 50 mM Li did not affect the quenching response at all. These results suggest that H may compete for the Li-binding site.

Inhibition of the Mutant F(1)F(0)-ATPase Activity by Li

The ATPase activity of A60E61S62T63 mutant membranes was inhibited by 50 mM LiCl when assayed in the HMK buffer at pH 8.5, whereas the activity of wild type and the A60E61A62T63 mutant was not (data not shown). The Li effects were studied in detail using membranes from cells overexpressing F(1)F(0), where the mutant membrane ATPase activity was higher and more easily measured. Strains carrying plasmids coding the entire unc operon, with either the wild type or the A60E61S62T63 mutant uncE gene, were constructed in the appropriate DeltanhaA, DeltanhaB host strain. The newly generated diploid wild type and A60E61S62T63 mutant strains exhibited three times more ATPase activity than their monoploid counterparts.

The effects of the Li on the ATPase activities of membranes prepared from the overproducing diploid cells are shown in Fig. 4. Li significantly inhibited the ATPase activity of A60E61S62T63 mutant membranes. Half-maximal inhibition was observed at 5-10 mM LiCl and 90% inhibition at 50 mM LiCl. ATPase activity was completely abolished by 100 mM Li (not shown). Mutant or wild type membrane ATPase activity was little affected by 50 mM NaCl. The wild type membrane ATPase activity was negligibly affected by Li at concentrations up to 50 mM. Li and Na also have negligible effects on the ATPase activity of purified F(1) in HMK assay buffer (data not shown), providing further support that the Li inhibition is mediated through the F(0) sector.


Figure 4: ATPase activity of A60E61S62T63 mutant membrane is inhibited by Li. Membranes (50 µg of protein) were diluted into 1.0 ml of HMK buffer, pH 8.5, containing LiCl (circle) or NaCl (Delta) at the concentration indicated, replacing a portion of the KCl. The ATPase activities were assayed at 25 °C after addition of Tris-[-P]ATP to 1 mM.



The inhibition of A60E61S62T63 membrane ATPase activity by Li is also pH dependent as shown in Fig. 5. Strong inhibition by Li was observed in the alkaline pH range but only moderate inhibition at neutral pH. The results again suggest that Li and H may compete for the same binding site. The Li effects on ATPase activity and ATP-driven H translocation show a similar dependence on pH. ATP-driven quenching of ACMA appears to be less sensitive to Li inhibition, but this is probably because ATPase activity can be inhibited substantially without significant reduction of the quenching response (Miller et al., 1990; Zhang and Fillingame, 1994). This rationale likely accounts for the observed Li inhibition of ATPase activity at pH 7.0, but lack of effect on ATP-driven quenching at the same pH.


Figure 5: Comparison of Li inhibition of A60E61S62T63 membrane ATPase activity at different pH values. Membranes (50 µg of protein) were diluted into 1.0 ml of HMK buffer at the pH and LiCl concentration specified. The other conditions are as described in Fig. 4.



Li Inhibition of Passive Fo-mediated H Transport

If Li inhibits ATPase coupled H translocation by A60E61S62T63 membranes via binding in F(0), we might expect that F(0)-mediated H transport should also be inhibited by Li. Wild type and A60E61S62T63 F(0) were isolated from membranes of the overproducing unc diploid strains with chromosomal DeltanhaA, DeltanhaB and the F(0) reconstituted into liposomes. H transport into K-loaded proteoliposomes was determined by quenching of ACMA fluorescence on addition of valinomycin. The rate of H uptake by A60E61S62T63 F(0) proteoliposomes was inhibited by 37% with 25 mM LiCl and 43% with 50 mM LiCl in the assay buffer (Fig. 6B). On average, the rate of H uptake decreased 50 ± 8% with 50 mM LiCl in five other independent experiments, whereas 50 mM NaCl had no effect on the rate of H uptake. A more rapid rate of H uptake was observed with wild type F(0) when the same amount of F(0) was used in the reconstitution, and this uptake was unaffected by either 50 mM LiCl or 50 mM NaCl (not shown). To test whether the Li inhibition observed with A60E61S62T63 proteoliposomes was due simply to the lower transport rate, proteoliposomes containing less amounts of wild type F(0) were prepared such that the rate of H uptake equaled that of the A60E61S62T63 proteoliposomes. Under these conditions (Fig. 6A), 25 and 50 mM LiCl decreased the rate of H uptake by wild type F(0) proteoliposomes by 6 and 16%, respectively. NaCl at 25 and 50 mM was without effect.


Figure 6: Effect of Li and Na on pH gradient-driven quenching of ACMA fluorescence by F(0)-containing proteoliposomes. K-loaded proteoliposomes (0.33 mg of phospholipid) were diluted into 3.2 ml of assay buffer, pH 8.4, containing LiCl or NaCl at the concentration indicated. ACMA was added to 0.3 µg/ml and the fluorescence quenching initiated by addition of valinomycin to 10 nM to generate a K diffusion potential. SF6847 was added at 0.3 µM. DCCD in 16 µl of ethanol was added to the diluted liposomes at a final concentration of 50 µM and the mixture incubated 10 min at 25 °C before the addition of ACMA and valinomycin.




DISCUSSION

We have generated an E. coli F(1)F(0)-ATPase that binds Li by replacement of 4 residues in subunit c with residues found in the equivalent positions of P. modestum subunit c. These substitutions include the DCCD-reactive Asp and the immediately surrounding residues. Na has no effect on the A60E61S62T63 enzyme. Li binds to the E. coli enzyme with an affinity close to that reported for the P. modestum enzyme (Kluge and Dimroth, 1993a). The K(i) for Li inhibition of the E. coli A60E61S62T63 ATPase was 5-10 mM at pH 8.5. In both the P. modestum and E. coli enzymes, Li and H appear to compete for the same site. ATP-driven H transport and ATPase activity for the E. coli A60E61S62T63 enzyme gradually decreased as the Li concentration was increased, and the inhibition by Li was reversed by increasing H concentration.

Our results show that Li inhibits both ATP-driven H uptake and valinomycin induced H influx into F(0)-containing liposomes. ATPase-coupled H transport and ATPase activity seem to be somewhat more sensitive to Li inhibition. This could indicate that Li binds preferentially to a form of the carrier in the active transport cycle. The form of the carrier mediating passive H transport may differ from the forms of the carrier in the active transport cycle, as was discussed previously (Fillingame, 1990).

In other experiments (not shown), we were unable to demonstrate a Li effect on H efflux from F(1)-stripped inverted membrane vesicles. In these experiments, H transport by F(0) was estimated by the extent of dissipation of a DeltapH (acid interior) established by electron transport coupled H transport (for method, see Zhang and Fillingame, 1994). These measurements were done under conditions where the membrane potential was dissipated. The explanation for lack of a Li effect may relate to the observations of Kluge and Dimroth(1992) that Na or Li inhibition of Delta-driven H influx into F(0) liposomes is dependent upon the side to which these ions are added. H uptake was abolished by ion addition to the outside of the proteoliposomes, whereas the same concentration of ion within the liposomes had no effect. To bind ion, the inhibitory site may have to move to the positively charged side of the membrane. The ion-binding site can obviously move to the outside of these vesicles, in the absence of a membrane potential, if F(1) is bound and ATP present in catalytic sites.

All our experiments were performed with membranes prepared from a DeltanhaA, DeltanhaB background. This proved to be necessary because both Li and Na do reduce the ATP-driven quenching response of mutant membranes showing low ATPase activity if the membranes have active antiporters. For example, 10 mM Na or 10 mM Li significantly reduced the ATP-driven ACMA quenching responses of both the A60E61S62T63 and A60D61S62T63 mutant membranes, when the mutant alleles were expressed in the AN346 background. We attribute this to Na/H antiporter activity that is also insensitive to 0.5 mM amiloride (Taglicht et al., 1991).

All four substitutions in the A60E61S62T63 subunit c proved necessary for Li binding. The V60E61S62T63, A60D61S62T63, and A60E61A62T63 subunit c mutant enzymes were active but were not inhibited by Li. The side chains of both Ala and Thr in the Li binding A60E61S62T63 enzyme are smaller than those of Val and Ile in the wild type protein. They may act in a supporting structural role by providing a more flexible conformation in the Li binding cavity. On the other hand, a polar residue appears to be required at position 62. We suggest that the Li-binding site could be formed, at least in part, by the Glu and Ser side chain oxygens, with perhaps the Glu peptide carbonyl oxygen acting as an additional liganding group (Fig. 7). A water molecule may also contribute to the Li binding since most of metal-binding sites contain one or two water molecules (Toney et al., 1993; Badger et al., 1994). The experiments of Kluge and Dimroth(1994) indicate that P. modestum subunit c binds Na in the absence of other F(0) subunits. We of course do not know whether the binding site is formed in a single subunit c or by a multimer.


Figure 7: A possible conformation of the Li binding site in a monomer of subunit c. The polypeptide backbone from a GluSer sequence in a normal alpha helix was used as a template to generate the structure. The side chain conformations in this structure were minimized by a combination of molecular dynamics and mechanics with 1.9-2.1 Å distance restraints for Na/Li binding to the two Glu carboxylate oxygens and the Ser oxygen. Li was then placed at an optimal distance (1.9-2.0 Å) within that structure. Hydrogen atoms are not shown.



Na has no effect upon and appears not to bind to the A60E61S62T63 subunit c modified E. coli enzyme. This may relate to several aspects of ion binding. The ionic diameter of Na (approximately 1.90 Å) is larger than that of Li (approximately 1.30 Å) (Hille, 1990). It is well known that cavity size of a metal-binding site is in good match to the ionic diameter of the cation most strongly bound, as shown for example by the relationship between the structure of crown ethers and their ability to complex various cations, i.e. 18-crown-6 has a high affinity for K, 15-crown-5 for Na, and 12-crown-4 for Li (Weber and Vogtle, 1981). Conceivably, additional small changes in the cation binding cluster geometry, by introduction of smaller or more flexible residues around position 61, might enable Na to bind to the protein. A second reason for absence of Na binding could be a requirement for an extra liganding group. An example is found in the structural analysis of dialkylglycine decarboxylase (Toney et al., 1993). Dialkylglycine decarboxylase specifically requires K for activity. However, in the absence of K, the smaller Na can fit tightly in the binding site with coordination to five oxygen ligands. With the larger K (ionic diameter: 2.66 Å) bound, the cation binding cavity diameter enlarges by about 0.8 Å, and the number of coordination oxygens increases from five to six by enclosing an additional hydroxyl group to the cation coordination sphere. In the case of subunit c, the A60E61S62T63 enzyme may lack a liganding group necessary for Na coordination, or because of additional structural barriers, such a liganding group may not be able to approach the cation bind site. The Pro and Gln replacements might provide the liganding groups in the P. modestum enzyme.

Coincident with this work, the sequence of the 16-kDa proteolipid subunit of the Na translocating ATPase of Enterococcus hirae was published (Kakinuma et al., 1993), and it shows a Thr residue next to the proposed DCCD reactive Glu. Val and Gly are found at this position in other ``V-type'' 16-kDa subunits. The side chain oxygens of Glu and Thr may contribute at least some of liganding groups in the Na-binding site of this enzyme.

In summary, these results demonstrate that the residues around the DCCD-reactive carboxyl of subunit c at least partially determine the cation specificity of F(0). We suggest that a X-Glu-Ser-Y or X-Glu-Thr-Y sequence may provide a general structural motif for monovalent cation binding and that the flexibility provided by residues X and Y will prove crucial to this structure. A definition of the P. modestum and E. hirae Na-binding sites will clearly have to await direct mutagenesis experiments with these enzymes. In the case of P. modestum F(1)F(0), we would now predict that a SerAla mutation in subunit c will result in loss of Na pumping with retention of H pumping.


FOOTNOTES

*
This study was supported in part by United States Public Health Service Grant GM23105 from the National Institutes of Health and by a grant from the Human Frontier Science Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a gift from the Lucille P. Markey Charitable Trust to the University of Wisconsin Medical School.

To whom correspondence should be addressed: Dept. of Biomolecular Chemistry, 587 Medical Sciences Bldg., University of Wisconsin-Madison, Madison, WI 53706. Tel.: 608-262-1439; Fax: 608-262-5253.

(^1)
The abbreviations used are: DCCD, dicyclohexylcarbodiimide; ACMA, 9-amino-6-chloro-2-methoxyacridine; bis-tris-propane, 1,3-bis-[tris(hydroxymethyl)methylamino] propane; Tricine, N-tris(hydroxymethyl)methylglycine.

(^2)
The unc DNA numbering system corresponds to that used by Walker et al.(1984).

(^3)
We will subsequently identify multiple mutants by referring to the final residue at each position with mutated residues underlined.


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