©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Genetic Evidence for Two Sequentially Occupied K Binding Sites in the Kdp Transport ATPase (*)

(Received for publication, November 14, 1994; and in revised form, December 19, 1994)

Ed T. Buurman(§)(¶) Ki-Tae Kim(¶)(**) Wolfgang Epstein (§§)

From the Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Substrate binding sites in Kdp, a P-type ATPase of Escherichia coli, were identified by the isolation and characterization of mutants with reduced affinity for K, its cation substrate. Most of the mutants have an altered KdpA subunit, a hydrophobic subunit not found in other P-type ATPases. Topological analysis of KdpA and the locations of the residues changed in the mutants suggest that KdpA has 10 membrane-spanning segments and forms two separate and distinct sites where K is bound. One site is formed by three periplasmic loops of the protein and is inferred to be the site of initial binding. The other site is cytoplasmic. We believe K moves from the periplasmic site through the membrane to the cytoplasmic site where it becomes ``occluded,'' i.e. inexchangeable with K outside the membrane. Membrane-spanning parts of KdpA probably form the path for transmembrane movement of K. The kinetics of cation transport in the mutants indicate that each of the two binding sites contributes to the observed K for cations as well as to the marked discrimination between K and Rb characteristic of wild-type Kdp. Energy coupling in Kdp, mediated by the KdpB subunit, is performed by a different subunit from the one that mediates transport.


INTRODUCTION

Active transport systems are enzymes that convert chemical or physical energy to transmembrane movement rather than to the production of a chemical product. Like other enzymes, transport systems have sites where the transported substrate is recognized and bound. The substrate site is accessible to the side from which uptake occurs. Upon binding of substrate, transport systems undergo a conformation change which seals off the substrate from the presenting side. The substrate enters a path across the membrane and is ultimately released into the compartment on the other side. The conformational changes associated with transport are coupled to both energy and substrate binding; uncoupled movement does not occur(1) .

Active transport systems, like enzymes generally, are inherently reversible. Transport in the reverse direction from that normally observed has been demonstrated for several transport systems when substrate gradients and energy parameters are suitably adjusted(1, 2, 3) . Therefore, transport systems must have a substrate binding site accessible from each side of the membrane. The two binding sites implied by reversibility of transport may be separate and distinct, with one on each side of the membrane. Kinetic models of active transport systems suggest dual substrate binding sites(1, 2) . Alternatively, reversible transport could be produced by a single binding site that, like the two-faced Janus, is exposed to either the outside or the inside as determined by the conformation of the transport system. This single site would have two functionally different states even though formed by the same part of the protein, because the conformation of the protein differs depending on which way the site ``faces.''

Few studies have addressed the question of binding sites in transport systems directly. The three-dimensional structure of no transport system is known. There is only limited evidence as to the regions of a transport system that bind substrate. An analysis of a large number of site-directed mutations of the Ca-ATPase of sarcoplasmic reticulum has implicated residues in predicted membrane spans as determinants of the enzyme's affinity for Ca(4) . These results have been interpreted in terms of the one-site Janus model(5) .

We here report a study of substrate binding by the Kdp system, a P-type transport ATPase of Escherichia coli that accumulates K in the cell and has high affinity and specificity for K. P-type transport ATPases are found in all biological kingdoms and mediate transport of a variety of cations(6) . All P-type ATPases have a homologous, large membrane-bound subunit, ranging in size from about 70 to 130 kDa, and form an aspartyl-phosphate derivative of the large subunit. The aspartyl-phosphorylated derivative is an energetic intermediate in transport.

P-type ATPases differ in their substrates, in their directions of transport and in their subunit composition. Substrates include the monovalent cations H, Na, and K and the divalent cations Ca, Mg, Cd, and Cu. Some of these enzymes export ions from the cytosol, others mediate transport into the cytosol, and several mediate both processes in a concerted reaction. Examples of the latter include the electrically neutral gastric K,H-ATPase and the electrogenic Na,K-ATPase. Some of these enzymes have only a single large subunit, and others have two, while Kdp is unique as the only P-type ATPase with three protein subunits. Small peptides associated with many of these ATPases may modulate these enzymes, although they do not seem to be necessary for their activity(7, 8, 9, 10) .

The largest subunit of Kdp (for review, see (11) ), the 72-kDa KdpB protein, is the homolog of the large subunit of other P-type ATPases. Neither of the other subunits, the 59-kDa KdpA protein or the 20.5-kDa KdpC protein, has homologs in other P-type ATPases. KdpA is very hydrophobic with many predicted membrane-spanning segments. KdpC is predicted to have only a single membrane-spanning segment. All three subunits are required for activity, and associate in a structure that is stable to solubilization with non-ionic detergents. KdpF, a hydrophobic 29-residue peptide, may also be a part of Kdp. This peptide is encoded in the same operon as are the three large subunits, and it is known to be expressed(11) . The Kdp complex has been purified to near homogeneity(12) , and ATP-driven K transport has been observed when the Kdp complex is reconstituted in vesicles(13) .

Kdp is one of three separate and saturable K uptake systems found in E. coli. The other two systems, Trk and Kup (for review see (14) ), have affinities for K in the vicinity of 1 mM. In wild-type strains, loss of Kdp activity results in only a modest increase in the concentration of K needed to achieve rapid growth. When all three systems are lost, a medium concentration of about 25 mM is required to achieve half-maximal growth rate, and growth below about 15 mM is so slow as to be negligible. When only Kdp is present, growth is rapid even in medium containing only µM concentrations of K because Kdp has very high affinity for K.

We have exploited the large effects of Kdp on growth in strains lacking Trk and Kup to isolate Kdp mutants that are competent for transport but have markedly reduced affinity for K. After mutagenesis, penicillin selection killed cells with parental wild-type Kdp activity that grow at 0.1 mM K. Recovery from the penicillin treatment was in medium containing 5 or 10 mM K, a medium in which strains lacking Kdp activity do not grow.

The selection yielded 37 independent mutants that fit the growth properties of the selection. All of the mutants harbor a Kdp system with markedly reduced affinity for K. 33 of the mutants alter the KdpA subunit. The amino acid residues altered by the mutations are clustered in four regions of KdpA. Topological analysis indicates that three of the clusters are in periplasmic loops and one of the clusters is in a relatively hydrophobic but cytoplasmic loop of KdpA. From these results we infer that Kdp has two binding sites for K, both formed by the KdpA protein: an initial binding site in the periplasm and a site in the cytoplasm where K is subsequently bound and which may represent the site of K occlusion.


EXPERIMENTAL PROCEDURES

Isolation, Cloning, and Sequencing of Mutations

Mutants were isolated after ultraviolet mutagenesis of strain TK2240 (Fthi lacZ rha nagA trkA405 trkD1) followed by penicillin selection for kdp mutants with reduced affinity for K as described(15) . Only a single mutant with a given phenotype from each of 18 separately mutagenized lots of cells was retained for further study. The mutations were transferred to strain TK2247 (a nadA derivative of TK2240) by P1-mediated transduction with selection for nad. In most cases the mutant phenotype was expressed by about 15% of the transductants, as expected from the linkage of kdp to nadA. A few mutants seemed to harbor kdp null alleles plus a mutation that partially corrected the Trk defect; these were discarded. A few mutations were cloned into derivatives of pBR322 by shotgun cloning of EcoRI-cut chromosomal DNA; most were cloned with Mini-Mu plasmid pEG5005(16) . Clones carrying the kdp region were selected by complementation of strain TK2205 (a Delta(kdpABC)5 derivative of TK2240) to growth at 5 mM K, or in a few cases of strain TK2230 (a kdpB30 derivative of TK2240) to growth at 0.1 mM K, which required only expression of a wild-type kdpB gene. The 4.9-kb (^1)EcoRI fragment that carries kdpABC was cut from the Mini-Mu plasmids and inserted into the EcoRI site of pJD100, a EcoRV-PvuII deletion derivative of pBR322.

Mutations were assigned to genes by complementation and/or recombination with kdp deletions(15) . Finer mapping was done by recombination with plasmids that divided kdpA into five segments and in a few cases by recombination with M13 clones used in the initial sequence analysis of the region(17) . Five of the mutations were sequenced by the dideoxy method (18) with Sequenase (U. S. Biochemical Corp.) after cloning a 100-400-base pair segment into M13mp7 or M13mp8. The rest were sequenced after cloning into pJD101 by double-stranded sequencing (19) with Sequenase and primers that hybridize within 200 base pairs of the mutations. Four primers that direct sequencing toward the C terminus were used: Af, bases 284-297; Bf, 589-603; Cf, 967-979; Df, 1311-1325. Two primers that direct sequencing toward the N terminus were used: Ab, 403-417; Cb, 1228-1242 (numbering of bases is that of (17) ).

Kinetic Characterization of the Mutants

The dependence of growth rate at 37 °C on medium K and initial rates of uptake of K and of Rb by K-depleted cells at 30 °C were measured as described (15, 20) . All data for Rb and a few for K are from a single experiment; most of the data for K represent the average of at least duplicate experiments. Transport kinetics are based on uptake rates at three or more cation concentrations. For the transport assays, mutants were grown at a K concentration that resulted in a growth rate between 50 and 60% of that at high K, since this results in a nearly full induction of Kdp in the mutants(21, 22) . In a few mutants, the ability of Cs to inhibit uptake of K was also tested, with a K concentration at or near the K(m) and Cs concentrations of 10 or 50 mM. Transport rates are expressed per gram (dry weight), determined from turbidity measurements and a calibration curve.

Construction of Protein Fusions

The plasmids used to construct fusions are shown in Fig. 1. Protein fusions were constructed in the low copy number plasmids pPAB203, pPAB307, or pPAB404 where expression is under tac promoter control to minimize possible problems due to high level expression of the fusions. pPAB203 was constructed by inserting the PstI fragment from plasmid pCH2 (23) containing the phoA gene without its promoter, signal sequence or first few amino acids, into the PstI site of pJFK118EH (identical to pJF118EH, (24) , but with the multicloning site of pUC19). The resulting plasmid was cut with HindIII, treated briefly with BAL31 nuclease to remove the distal PstI site, and then religated to generate pPAB203. Plasmids pPAB307 and pPAB404 are similar but used the PstI fragment from plasmids pCH39 and pCH40, respectively, to place the PstI site in different reading frames relative to phoA.


Figure 1: Plasmids used to make fusions of kdpA to phoA and to lacZ. Construction of these vectors is described under ``Experimental Procedures.'' A, phoA fusion vectors with PstI sites in all three reading frames. pPAB203 is shown, while pPAB307 and pPAB407 are identical except for the position of the multicloning site relative to the reading frame of phoA. Protein fusions were created when fragments of kdpA were inserted in the multicloning site. B, pKAB101 served as the PCR template to construct kdpA fragments with a distal PstI site. Shown diagrammatically are the locations of the common upstream primer on the left, and one example of a downstream primer that carries the PstI site used to create a fusion. C, pJLZ104 was used to convert phoA to lacZ fusions. The fusions to phoA were cut with EcoRI and DraI and inserted into pJLZ104 cut with EcoRI and SmaI to create fusions that contain about 60 residues of PhoA prior to the start of LacZ residues.



pKAB101 served as template for the polymerase chain reaction to construct kdpA fragments with a distal in-frame PstI site. The 2.29-kb BclI-HpaI fragment of pWE1001 (17) was inserted into pUC19 cut with BamHI and HincII to make pKAB101. The desired fragments were made by using linearized plasmid, a primer just upstream of the EcoRI site and a primer with a PstI site in the proper reading frame just past the desired fusion point with the Gen Amp kit and following the manufacturer's recommended conditions for the polymerase chain reaction (Perkin-Elmer Cetus). The fragments obtained were cut with KpnI and PstI and ligated into pPAB203 that had been cut with the same enzymes.

pJLZ104 was used to convert phoA to lacZ fusions. The 4.85-kb SmaI-NruI fragment of pRS414 (25) was cloned into M13mp18 (26) that had been digested with SmaI and HindII. The resulting construct was digested with EcoRI and HindIII and cloned into similarly digested pJFK118EH to make pJLZ104. lacZ fusions were constructed by digesting a phoA fusion cloned in pPAB203 (or pPAB307, or pPAB404) with EcoRI and DraI (which cuts early in phoA) and ligating the fusion fragment into pJLZ104 cut with EcoRI and SmaI. The lacZ fusions carry residues 14-73 of mature PhoA (residues 6-73 in the case of the fusion at residue 100) prior to joining LacZ at residue 9.

The phoA fusion at residue 100 was made by inserting the KpnI-XhoII fragment of pKAB101 into pPAB307 that had been digested with KpnI and BamHI. The phoA fusion at residue 350 was made by cloning the KpnI-NsiI fragment of pKAB101 into pPAB404 digested with KpnI and PstI. All fusions to phoA were confirmed by sequencing over the fusion joint in the backward direction, using as primer a 19-mer oligonucleotide that hybridized to the region of phoA corresponding to amino acid residues 35-41 of mature PhoA.

Enzymatic Assays

The phoA and lacZ fusions were maintained and studied in strains BW14892 (27) and XL-1-Blue (Stratagene, La Jolla, CA), respectively. Cells were grown in KML medium (per liter: tryptone, 10 g; yeast extract, 5 g; KCl, 10 g) containing ticarcillin (100 µg ml) in the presence of 1 mM isopropyl-1-thio-beta-D-galactopyranoside. When the cultures reached an OD of 0.3-0.4, cells were harvested and alkaline phosphatase and beta-galactosidase activities were determined according to Brickman and Beckwith (28) and Miller (29) , respectively.

Immunoblot Analysis of phoA Fusions

We prepared filters with bacteria proteins essentially as described by Siebers and Altendorf(30) . Cells were grown at 37 °C in KML medium containing 1 mM isopropyl-1-thio-beta-D-galactopyranoside, harvested by low speed centrifugation, washed in 0.1 M Tris-HCl (pH 7.6), and disrupted by boiling for 5 min in the presence of 2% sodium dodecyl sulfate. Total proteins were separated electrophoretically on 7.5% polyacrylamide-sodium dodecyl sulfate gels and transferred electrophoretically to nitrocellulose membranes. phoA fusions were detected as described by Mierendorf et al.(31) using a polyclonal anti-E. coli alkaline phosphatase antibody (Rockland, Gilbertsville, PA) and the Protoblot system (Promega, Madison, WI); blocking and washing were done with a solution containing 0.67 M NaCl, 50 mM Tris-HCl, pH 8, and 0.5% (w/v) nonfat dry milk.


RESULTS

Isolation and Characterization of Mutants

A total of 37 independent mutants were isolated as described above. The growth, genetic characteristics, and transport kinetics of the mutants are summarized in Table 1. We refer to these mutants as having a reduced affinity for K, meaning the apparent affinity as measured by the K(m) of transport, the concentration of K at which uptake is half-maximal. The selection, based on the growth properties, resulted in a series of strains in which the K(m) for K in transport increased to the range from 200 µM to over 50 mM, much higher than the value of 2 µM for wild-type Kdp.



A total of 19 different mutations was obtained; several were obtained more than once. Fifteen of the mutations, representing 33 independent mutants, altered the KdpA subunit, three altered KdpB, and one altered KdpC. The effect of a mutation on the maximum rate of transport depended on the subunit altered by the mutation. All mutations altering KdpA resulted in a V(max) that was at least 30% of the normal rate, while only mutant 57 of the four mutations that altered other subunits had this high a rate. The fact that most mutants selected to have lower affinity for K change the KdpA subunit implies that KdpA has a major role in determining affinity for K. Furthermore, mutations can markedly reduce affinity without much effect on V(max). The lower number of mutations affecting KdpB or KdpC suggests that there are fewer changes in those subunits that can markedly reduce affinity for K. Therefore, these two subunits probably have a lesser role in determining affinity for K than does the KdpA subunit.

Most of the mutations reduced affinity for Rb as well, but in a few cases the affinity increased (Table 1). Discrimination between K and Rb was retained but at a reduced level. The ratio of the K(m) values for these two ions fell from the 4000-fold difference seen in the wild-type to a ratio of 25 or less. In mutant 13 the ratio of the affinities was 1, indicating that this mutant had lost the ability to discriminate between these ions. The even larger Cs ion was able to inhibit K uptake in a few of the mutants so tested, suggesting Cs is a substrate of Kdp in those mutants. Uptake of Cs by wild-type Kdp is not detectable(32) .

Sequence Changes in the Mutants

The sequence analysis showed that the mutations in the kdpA gene were in four regions of the gene that encode residues 114-116, 223-232, 345-369, and 461-469 (Table 1). There is no simple correlation between the change in affinity and either the location or the nature of the change in amino acid residue. All of the residues altered, except for Gly-345, are in extramembraneous regions of the protein (Fig. 2). There are three changes from a neutral to an acidic residue, two changes of neutral to basic, and one from acidic to basic. In the other mutations, where one neutral residue replaces another neutral residue, there is a tendency for the new residue to be less polar.


Figure 2: Topology of the KdpA protein. Membrane-spanning regions have a length consistent with helical barrels, and extramembrane regions are shown as arbitrary loops. The membrane region is at the center, enclosed by dashedlines. The amino and carboxyl termini and every 50th residue are labeled. The sites of the affinity mutations of Table 1are circled. The region of homology with other transport ATPases is enclosed in a pentagon, at topright. Boxesenclose the points of protein fusions, whose activities are given in a box connected to the site of the fusion; PhoA is alkaline phosphatase activity, Z is beta-galactosidase activity. The enzyme activities, measured as described under ``Experimental Procedures,'' are averages of four determinations. The average standard deviation was 22% of the measured value for the alkaline phosphatase assays and 18% of the value for beta-galactosidase. As described in the text, fusions at residues 375 and 378 were also made in which Glu-370 was changed to Ala to test an alternate, 12-membrane domain model. Alkaline phosphatase activities were 4 ± 1 and 3.6 ± 0.4, and beta-galactosidase activities were 350 ± 20 and 286 ± 10, in the fusions at residues 375 and 378, respectively. Control alkaline phosphatase activity, measured under the same conditions with plasmid pPAB203, was 4 ± 1 unit; control beta-galactosidase activity with plasmid pJLZ104 was 5 ± 6 units.



Transition and transversion mutations are almost equally represented among the sequenced mutations, similar to the distribution of ultraviolet light induced mutations in lacI(33) . Two examples of a double mutation were obtained. Mutants 78 and 86, whose primary lesion and kinetics are like those of 15, also had a change of C to A at base 682 to replace Leu-228 with Ile. We believe this is a functionally silent change because the growth and transport properties of these two mutants do not seem to differ from those of mutant 15, which retains the wild-type Leu-228.

Topology of KdpA

We examined the topology of KdpA in order to determine the locations of the four clusters identified by the mutants. Most protein structure analysis programs based on hydrophobicity predict that KdpA will have 12 membrane spans with 11 extramembraneous regions(34, 35) . To test this model we made protein fusions to phoA, the structural gene for alkaline phosphatase. Alkaline phosphatase is active only when placed in the periplasm, and its location in a fusion appears to be dictated by the site of fusion(23, 36) . Fusions were created in each of the predicted extramembrane segments, with two fusions made in each of three of the segments, using the plasmids diagrammed in Fig. 1. The analysis of the fusions suggests the topologic model of KdpA presented in Fig. 2. That figure also shows the residues altered by the mutations of Table 1, and the locations and activities of the protein fusions.

The fusions to alkaline phosphatase had either very low activity, often not different from the control, or relatively high activity. Low activity is consistent with fusions to cytoplasmic regions, while high activity suggests fusion to externally exposed regions(36) . These results imply that KdpA has only 9 extramembraneous regions and 10 membrane spans as shown in Fig. 2. The one intermediate level of alkaline phosphatase activity is to residue 336 in a membrane span. Fusions to residues in membrane spans have intermediate levels of activity(37) .

The topologic model predicted by programs based on hydrophobicity includes two additional membrane spans encompassing residues 345-376 and 379-399. The low alkaline phosphatase activity of fusions at residues 375 and 378, which are cytoplasmic in Fig. 2but would form a small periplasmic loop in the 12-membrane span model, are not consistent with the 12-span model. We considered the possibility that the Glu residue at 370 might have prevented proper insertion of the fusions at residues 375 and 378. Glu-370 is in the middle of membrane span VIII of the 12-membrane span model. Aberrant localization of fusions has been observed in analogous situations in studies of other membrane proteins(37, 38) . When Glu-370 was changed to Ala in the fusions, there was no increase in the low levels of alkaline phosphatase activity of fusions at these sites (Fig. 2, legend). Thus, misplacement of the region due to Glu-370 can be excluded.

Low activity of some of the fusions could be due to poor expression of the fused protein. Therefore fusions to lacZ (see ``Experimental Procedures'') were constructed at all sites with low alkaline phosphatase activity. At all of these sites, the lacZ reporter gene was expressed at a relatively high level, consistent with good expression at sites where alkaline phosphatase activity was low (Fig. 2). The expression of beta-galactosidase from fusions at 375 and 378 remained relatively high when Glu-370 was replaced by Ala. The fusion at residue 336, predicted to be in the membrane, had the lowest activity of any of the lacZ fusions, consistent with the predicted location(37) .

Since the stability and expression of fusions to phoA might differ from those to lacZ at the same sites, we examined expression of the phoA fusions directly by immunoblot analysis. The results indicate that very low levels of alkaline phosphatase activity cannot be attributed to low stability or low expression of the fusions. When cells were harvested in the logarithmic phase of growth, a band of the expected size was detected for all phoA fusions except the one to residue 162 (Fig. 3A). The level of expression varied somewhat, as can be seen in the figure. In other immunoblots in which variable amounts of extracts were compared, the fusions with the lowest level of expression were present at over 25% of the level of highly expressed fusions (data not shown). All of these fusions proved to be unstable. In samples from cells grown into the stationary phase, the majority of the fusions showed few if any PhoA-specific bands, while a minority showed good reactivity. The most intense PhoA-specific bands had a higher electrophoretic mobility, showing that they represent degradation products (Fig. 3B).


Figure 3: Immunoblot analysis of kdpA::phoA fusions. Samples of 100 µg of total protein were separated by electrophoresis and proteins reacting with an antibody to E. coli alkaline phosphatase were stained as described under ``Experimental Procedures.'' Bands seen in all lanes and especially prominent in panelB are due to other reactivities of the antibody used. Numbers identify the last residue of KdpA in each of the fusions. Only parts of control lanes are visible at the left. A, extracts from cells harvested in logarithmic phase. The lane for fusion 161 in panelA was obliterated in transfer; when separated on another gel no immunoreactive material different from the control was seen. B, extracts from cells harvested in the stationary phase.



We noted a correlation between location of a fusion and its stability; fusions to periplasmic regions of KdpA were relatively stable and were expressed at a higher level than were any of the fusions to internal regions. The only exceptions were the fusions at residues 61 and 162; the former has only the first membrane domain of KdpA and was quite stable, while the fusion at residue 162 was so unstable it was not seen. The fusion at Ala-336 in the membrane was among the more stable.


DISCUSSION

The data presented suggest the following steps in the transport of K by Kdp. First, K is bound to a site in the periplasm, a site formed by periplasmic loops 1, 2, and 4 of KdpA. K bound in this site is in diffusional equilibrium with the ion in the periplasmic space. A subsequent conformational changes seals off the site and allows K to move across the membrane through a channel formed largely or solely by membrane spans of KdpA, to arrive at a second binding site in a cytoplasmic part of the KdpA protein. A further conformational change releases K into the cytoplasm to complete the transport cycle. This transport cycle is suggested by the topology of KdpA and the locations of residues that we believe are in K binding sites.

The topologic model of KdpA shown in Fig. 2is supported by the protein fusion data and by the distribution of charged residues in Kdp. In membrane proteins of bacteria, basic amino acid residues are markedly underrepresented in periplasmic regions whereas acidic residues are more evenly distributed(39) . The model of KdpA fits this rule, since only 16% (4/24) of the Arg + Lys residues of KdpA are in external regions, whereas 46% (11/24) of the Asp + Glu residues are external. The only reason to consider other topologic models is the prediction, based on hydrophobicity(34) , of two additional membrane spans encompassing residues 345-376 and residues 379-399. Since no experimental data support this alternative structure, we conclude that three of the clusters identified by the mutants are periplasmic and one cluster is cytoplasmic.

The ends of most of the membrane spans are marked by an abrupt change from a region of hydrophobic residues to one very polar residue or two polar residues (Fig. 2). The cytoplasmic end of membrane span IX is not so clearly defined. Following Arg-493, shown ending span IX in Fig. 2, there are 16 hydrophobic residues. Our belief that these are in the cytoplasm is supported by the homology of a 10-residue part of this region (Fig. 2, pentagon) with a similar sequence in the large subunit of other P-type ATPases (Table 2). Since the corresponding region of the other ATPases is believed to be cytoplasmic(49, 50, 51) , we have drawn membrane span IX to end at residue 493 so that this region is cytoplasmic. Lys-510 at the end of this region of homology is widely conserved among these ATPases, but it does not have an important function in the Ca-ATPase from rabbit sarcoplasmic reticulum. Changing the corresponding Lys-728 to Ala had no discernible effect on that enzyme(52) .



Our topologic analysis of KdpA, done in the absence of other Kdp subunits, followed the approach used in the analysis of the two integral membrane proteins of the maltose transport system of E. coli. Protein fusions to MalF or MalG were analyzed in the virtual absence of the other subunit(53, 54, 55) . At many sites on a protein, bulky fusions would interfere with assembly and thereby create artifactual structures that could confound the analysis. KdpA does not require either of the other subunits to become inserted in the membrane (56) . KdpB does not seem to play a role in assembly of KdpA, since it is not associated with KdpA if the smaller KdpC subunit is not made. (^2)These considerations suggest that assembly of KdpA alone creates a structure similar or identical to that in the intact Kdp complex.

Our analysis implies that the residues in KdpA altered in the mutants are either in or very near sites for K binding. The marked change in K(m) for transport, the altered discrimination between K and its congeners (Table 1), and the clustering of the mutations support this interpretation. If the residues implicated here are not in binding sites, they must alter binding sites by an allosteric effect, by a conformational change transmitted from the site of the mutational change to the binding site. Conformational changes sufficient to alter a binding site ought to have other effects such as altering the rate of transport. However, since almost none of the mutations resulted in a marked change in rate, all but small conformational changes would seem excluded. A second argument against an allosteric interpretation of the mutations is that it is hard to understand why mutations in binding sites would not have been obtained. Changes in a function are most easily produced by changes in the site responsible for the function, rather than indirectly by a conformational change due to alterations elsewhere in the protein.

Three of the mutations altering the other subunits are consistent with allosteric effects on transport. Mutant 50 is unstable, while mutants 17 and 49 have very low rates of transport (Table 1), all indications of significant conformational changes. Only mutant 57 of those affecting other subunits has a rate of transport not much different from that of the wild-type. We believe these mutants alter transport through allosteric effects, but they could act directly at the cytoplasmic site in KdpA because because both KdpB and KdpC have large cytoplasmic domains. Neither subunit is likely to interact with a periplasmic site because neither has a significant periplasmic domain (11) .

The importance of the residues in the periplasmic regions of KdpA is suggested by their conservation in the KdpA protein of Clostridium acetobutylicum.(^3)This Gram-positive spore forming anaerobe is only distantly related to E. coli. Its DNA has a GC content of only 22-25% compared with 50% for E. coli. Inspite of the large evolutionary distance separating the two species, 41% of the residues of the clostridial kdpA gene are identical to those of the E. colikdpA gene. All 9 residues in the periplasm identified by our mutants are conserved in the clostridial sequence. By contrast, only 2 of the 4 residues identified in the cytoplasmic binding site are conserved.

The cytoplasmic binding site may represent the site where K becomes ``occluded'' during transport. In both the Na,K-ATPase (51, 57) and the H,K-ATPase(58) , binding of K to the enzyme results in trapping stoichiometric amounts of the cation in a form that is only slowly exchangeable with the external solution. Since all P-type ATPases examined for occlusion exhibit this property, occlusion of K by Kdp in the process of transport can be inferred even though not yet demonstrated.

The location of binding sites in KdpA on both sides of the membrane leads one to believe that K crosses the membrane through a path formed largely or exclusively by several of the membrane spans of KdpA. One of the affinity mutations is in span VII, so at least the distal part of this span is on the path. The fact that another affinity mutation alters a residue adjacent to the beginning of span IX suggests the latter might also be part of the path.

The prototypic structure of cation binding sites comes from studies of ionophores (for review see (59) ). The cyclic and rather rigid depsipeptide valinomycin binds K and Rb in octahedral symmetry by ion-dipole interactions with backbone carbonyl groups, thereby replacing water of the hydrated ion. The more flexible channel-forming gramicidin has a dipolar inner surface, again provided by backbone carbonyl groups, to create a channel for monovalent cations with relatively little specificity. The general use of ion-dipole rather than ion-ion bonding is reflected in the structure of monovalent cation binding sites in soluble proteins. In the Tl derivative of trypsin (60) and in the monovalent cation binding site of dialkylglycine decarboxylase(61) , ion-dipole interactions predominate, although in each protein one of the interactions is with a carboxylate group. In neither of these binding sites is there much specificity for the ion. Specific ion effects in dialkylglycine decarboxylase result from the alteration of enzyme conformation when Na rather than K is bound. The cation binding sites in these soluble proteins, like such sites in KdpA, are formed by groups of residues that are separated in the linear sequence of the protein.

Cation binding sites in other K transporting enzymes such as the Na,K-ATPase remain elusive(51) . Chemical modification of some residues suggested that they have a role in binding. However, site-directed mutagenesis has not generally confirmed these results. Trypsin treatment of the enzyme results in a complex that retains all but 2 of the 10 membrane spans of the alpha subunit as well as parts of the beta subunit. The trypsin-treated enzyme can occlude K(62) . This result was interpreted to suggest that K occlusion involved segments near the C terminus of the alpha subunit. Similar results have been reported for trypsin-treated H,K-ATPase(58) .

Substrate binding of divalent cations by transport systems has been studied only in the Ca-ATPase of sarcoplasmic reticulum. Residues near the middle of membrane spans 4, 5, 6, and 8 have been implicated to bind Ca based on the analysis of site-directed mutants(63, 64) . Rates of transport were reduced in many of the mutants, and those with the largest change in affinity were usually devoid of transport activity. Affinity could be measured in mutants defective in transport if they retained a partial reaction of the ATPase, the formation of phosphoenzyme from ATP or from inorganic phosphate. The locations of the residues near the middle of membrane spans suggests the Janus model for the Ca-ATPase, a binding site in the membrane that is exposed to either the inside or outside by conformational changes(5) .

Do the very different locations of mutants that reduce substrate affinity of Kdp from those with this effect in the Ca-ATPase mean that substrate binding in these two P-type ATPases has no common features? We believe the limited types and numbers of mutants of the two systems analyzed to date make this conclusion premature. The mutants we isolated in Kdp are rather different from those studied in the Ca-ATPase. Our mutants had to have a large change in affinity to survive our screen. Mutants with smaller changes in affinity, typical of the mutants of the Ca-ATPase, are missing from our analysis. Conversely, a role of extramembraneous regions of the Ca-ATPase in substrate binding has not been excluded. We suggest that the analysis of neither system is complete. Both membrane and extramembrane regions probably contribute to substrate recognition and binding. A more complete analysis will be required to see if the apparent differences between these two enzymes reflect fundamental differences in substrate binding or whether they identify different aspects of similar mechanisms.

Kdp is unusual among P-type ATPases in having one subunit for energy coupling, KdpB, and another for substrate binding and transport, KdpA. This division of labor resembles that of bacterial transport systems, which have a periplasmic binding protein as well as ATP-binding peripheral membrane protein(s) and integral membrane proteins(65) . It has been suggested that Kdp was assembled in evolution from an ATP-coupling unit, the precursor of KdpB, that associated with a membrane porter, the precursor of KdpA, to couple transport to ATP hydrolysis(66) . That a membrane porter can be driven either chemiosmotically or by ATP is suggested by data for arsenite export protein ArsB encoded by a staphylococcal plasmid(67, 68) . When arsB is expressed in E. coli, efflux of toxic arsenite seems to be energized by the protonmotive force. A similar system encoded by plasmids found in E. coli has a structurally similar ArsB protein plus ArsA, an ATP-binding subunit. The system in E. coli mediates arsenite efflux driven by ATP. When the ArsA protein from E. coli is co-expressed with ArsB from staphylococci, resistance is higher than when ArsA is absent, suggesting that ArsA can serve to couple ATP to arsenite export by the staphylococcal ArsB protein.

Kdp may be a useful model for cation binding by P-type ATPases. The fact that other P-type ATPases have only a single subunit for energy coupling as well as for binding and transport of substrate suggests that Kdp may share only the basic mechanism of energy coupling to ATP with other enzymes of this group. However, at least some elements of substrate binding and transport in Kdp may resemble these events in other P-type ATPases.


FOOTNOTES

*
This work was supported by Grant GM22323 from NIGMS, National Institutes of Health. 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.

§
Present address: Dept. of Molecular and Cell Biology, University of Aberdeen, AB9 1AS Aberdeen, Scotland, United Kingdom.

These authors have contributed equally to this work.

**
Present address: Dept. of Biology, Koshin University, Pusan, 606-080, Korea.

§§
To whom correspondence should be addressed: University of Chicago, MGCB, 920 E. 58th St., Chicago, IL 60637. Tel.: 312-702-1331; Fax: 312-702-3172; wepstein{at}midway.uchicago.edu.

(^1)
The abbreviation used is: kb, kilobase(s).

(^2)
A. Siebers, K. Altendorf, and W. Epstein, unpublished data.

(^3)
A. Treuner, and P. Duerre, personal communication.


ACKNOWLEDGEMENTS

We thank Jean Daniel, Elizabeth Dorus, Joanne E. Hesse, and Sarah Sutton for expert assistance in different phases of this study; Peter Duerre for the unpublished clostridial Kdp sequence; Eduardo Groisman for suggestions on the use of Mini-Mu in cloning; and Andrew Wright, Robert Simons, Bernard Erni, and Barry Wanner for providing plasmids and strains.


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