Revisiting regulation of potassium homeostasis in Escherichia coli: the connection to phosphate limitation.

Two-component signal transduction constitutes the predominant strategy used by bacteria to adapt to fluctuating environments. The KdpD/KdpE system is one of the most widespread, and is crucial for K+ homeostasis. In Escherichia coli, the histidine kinase KdpD senses K+ availability, whereas the response regulator KdpE activates synthesis of the high-affinity K+ uptake system KdpFABC. Here we show that, in the absence of KdpD, kdpFABC expression can be activated via phosphorylation of KdpE by the histidine kinase PhoR. PhoR and its cognate response regulator PhoB comprise a phosphate-responsive two-component system, which senses phosphate limitation indirectly through the phosphate transporter PstCAB and its accessory protein PhoU. In vivo two-hybrid interaction studies based on the bacterial adenylate cyclase reveal pairwise interactions between KdpD, PhoR, and PhoU. Finally, we demonstrate that cross-regulation between the kdpFABC and pstSCAB operons occurs in both directions under simultaneous K+ and phosphate limitation, both in vitro and in vivo. This study for the first time demonstrates direct coupling between intracellular K+ and phosphate homeostasis and provides a mechanism for fine-tuning of the balance between positively and negatively charged ions in the bacterial cell.

Absolute quantification of the Kdp subunits of Escherichia coli by multiple reaction monitoring.

The sensor kinase/response regulator system KdpD/KdpE of Escherichia coli regulates the expression of the kdpFABC operon, encoding the high-affinity KdpFABC potassium (K(+) )-transport complex. Additionally, it has been suggested that the kdpDE operon itself is subjected to autoregulation by its gene products KdpD and KdpE. However, since kdpFABC and kdpDE expression has mainly been studied on the transcriptional level, accurate information on absolute amounts and the stoichiometric subunit composition of KdpFABC and KdpD/KdpE under K(+) -limiting and K(+) -nonlimiting growth conditions are lacking. In this study, we used highly sensitive mass spectrometric methods to quantify the amount of subunits of the Kdp(F)ABC complex and KdpD/KdpE. Data-dependent shotgun MS was used to assess protein coverage and accessible peptides. Absolute amounts of Kdp(F)ABC and KdpD/KdpE were quantified by targeted MRM analysis in the presence of corresponding heavy labeled standard peptides. Baseline synthesis of Kdp(F)ABC and KdpD/KdpE was found to be in the attomolar range under K(+) -nonlimiting conditions. Under K(+) -limitation, synthesis of Kdp(F)ABC (KdpA:KdpB:KdpC ratio of 1:1:1) was amplified more than 100-fold, whereas only a tenfold amplification of KdpD/KdpE (KdpD:KdpE ratio of 1:4) was observed. The results obtained provide a solid basis for follow-up studies on the dynamic regulation of the Kdp system.

The sensor kinase KdpD of Escherichia coli senses external K+.

The Kdp system of Escherichia coli is composed of the high-affinity K(+) transporter KdpFABC and the two regulatory proteins KdpD (sensor kinase) and KdpE (response regulator), which constitute a typical two-component system. The kdpFABC operon is induced under K(+) -limiting conditions and, to a lesser extent, under high osmolality in the medium. In search for the stimulus sensed by KdpD, we studied the inhibitory effect of extracellular K(+) on the Kdp system at pH 6.0, which is masked by unspecific K(+) transport at higher pH values. Based on KdpD derivatives carrying single aspartate replacements in the periplasmic loops which are part of the input domain, we concluded that the inhibition of the Kdp system at extracellular K(+) concentrations above 5 mM is mediated via KdpD/KdpE and not due to inhibition of the K(+) -transporting KdpFABC complex. Furthermore, time-course analyses of kdpFABC expression revealed that a decline in the extracellular K(+) concentration efficiently stimulates KdpD/KdpE-mediated signal transduction. In this report we provide evidence that the extracellular K(+) concentration serves as one of the stimuli sensed by KdpD.

Synthesis and conformational analysis of efrapeptins.

The efrapeptin family of peptide antibiotics produced by the fungus Tolypocladium niveum, and the neo-efrapeptins from the fungus Geotrichum candidumare inhibitors of F(1)-ATPase with promising antitumor, antimalaria, and insecticidal activity. They are rich in C(α)-dialkyl amino acids (Aib, Iva, Acc) and contain one ß-alanine and several pipecolic acid residues. The C-terminus bears an unusual heterocyclic cationic cap. The efrapeptins C-G and three analogues of efrapeptin C were synthesized using α-azido carboxylic acids as masked amino acid derivatives. All compounds display inhibitory activity toward F(1)-ATPase. The conformation in solution of the peptides was investigated with electronic CD spectroscopy, FT-IR spectroscopy, and VCD spectroscopy. All efrapeptins and most efrapeptin analogues were shown to adopt helical conformations in solution. In the case of efrapeptin C, VCD spectra proved that a 3(10)-helix prevails. In addition, efrapeptin C was conformationally studied in detail with NMR and molecular modeling. Besides NOE distance restraints, residual dipolar couplings (RDC) observed upon partial alignment with stretched PDMS gels were used for the conformational analysis and confirmed the 3(10)-helical conformation.

Influence of K+-dependent membrane lipid composition on the expression of the kdpFABC operon in Escherichia coli.

The membrane-bound sensor kinase KdpD and the cytoplasmic response regulator KdpE regulate the expression of the kdpFABC operon coding for the high affinity potassium uptake system KdpFABC in Escherichia coli. The signal transduction cascade of this two component system is activated under K(+)-limiting conditions in the medium, but is less sensitive to high osmolality. In order to test whether K(+) limitation affects membrane phospholipid composition and whether this change affects kdpFABC expression, we analysed the phospholipid composition of E. coli under these conditions. Our measurements revealed that there is an increase in the cardiolipin (CL) content during the exponential growth phase at the expense of the zwitterionic phospholipid phosphatidylethanolamine. The higher anionic phospholipid content occurs along with an increase of transcriptional activity of the cls gene coding for CL synthase. Furthermore, in vivo studies with E. coli derivatives carrying mutations in genes coding for enzymes involved in phospholipid biosynthesis revealed that the increase in the anionic lipid composition enhances the expression rate of the kdpFABC operon. Finally, we show that kinase activity of KdpD is stimulated in its native membrane environment by fusion with liposomes of anionic, but reduced with liposomes of zwitterionic phospholipids.

Constant c10 ring stoichiometry in the Escherichia coli ATP synthase analyzed by cross-linking.

The subunit c stoichiometry of Escherichia coli ATP synthase was studied by intermolecular cross-linking via oxidation of bi-cysteine-substituted subunit c (cA21C/cM65C). Independent of the carbon source used for growth and independent of the presence of other FoF1 subunits, an equal pattern of cross-link formation stopping at the formation of decamers was obtained.

Osmotic Stress.

Escherichia coli and Salmonella encounter osmotic pressure variations in natural environments that include host tissues, food, soil, and water. Osmotic stress causes water to flow into or out of cells, changing their structure, physics, and chemistry in ways that perturb cell functions. E. coli and Salmonella limit osmotically induced water fluxes by accumulating and releasing electrolytes and small organic solutes, some denoted compatible solutes because they accumulate to high levels without disturbing cell functions. Osmotic upshifts inhibit membrane-based energy transduction and macromolecule synthesis while activating existing osmoregulatory systems and specifically inducing osmoregulatory genes. The osmoregulatory response depends on the availability of osmoprotectants (exogenous organic compounds that can be taken up to become compatible solutes). Without osmoprotectants, K+ accumulates with counterion glutamate, and compatible solute trehalose is synthesized. Available osmoprotectants are taken up via transporters ProP, ProU, BetT, and BetU. The resulting compatible solute accumulation attenuates the K+ glutamate response and more effectively restores cell hydration and growth. Osmotic downshifts abruptly increase turgor pressure and strain the cytoplasmic membrane. Mechanosensitive channels like MscS and MscL open to allow nonspecific solute efflux and forestall cell lysis. Research frontiers include (i) the osmoadaptive remodeling of cell structure, (ii) the mechanisms by which osmotic stress alters gene expression, (iii) the mechanisms by which transporters and channels detect and respond to osmotic pressure changes, (iv) the coordination of osmoregulatory programs and selection of available osmoprotectants, and (v) the roles played by osmoregulatory mechanisms as E. coli and Salmonella survive or thrive in their natural environments.

The stoichiometry of subunit c of Escherichia coli ATP synthase is independent of its rate of synthesis.

Immunoblot quantitation of Escherichia coli ATP synthase isolated from atp wildtype and mutant cells, the latter comprising a reduced expression of the atpE gene coding for subunit c due to a point mutation within its Shine-Dalgarno sequence, suggested a variable stoichiometry of subunit c [Schemidt et al. (1995) Arch. Biochem. Biophys. 323, 423-428]. To study the c ring of the mutant strain and its stoichiometry in more detail, F O isolated from wildtype and mutant were investigated by quantitation, reconstitution, and cross-linking. Direct quantitation by staining with SYPRO Ruby revealed a reduction of subunit c in the mutant by a factor of 2 compared to F O subunits a and b. Rates of passive H (+) translocation correlated with the amount of subunit c present. Lower rates for mutant F O could be increased by addition of subunit c, whereas translocation rates remained constant by coreconstitution with nonfunctional subunit cD61G arguing against the presence of smaller c rings that are filled up with coreconstituted subunit c. Intermolecular cross-linking by oxidation of bicysteine-substituted subunit c ( cA21C/ cM65C) revealed an equal pattern of oligomer formation in wildtype and mutant also favoring a comparable subunit c stoichiometry. Cross-linking of membrane vesicles containing cysteine-substituted subunits a ( aN214C) and c ( cM65C) characterized the mutant F O preparation as a heterogeneous population, which consists of assembled F O and free ab 2 subcomplexes each present to approximately 50%. Thus, these data clearly demonstrate that the stoichiometry of the subunit c rings remains constant even after reduction of the synthesis of subunit c.

Reduction of turgor is not the stimulus for the sensor kinase KdpD of Escherichia coli.

Stimulus perception by the KdpD/KdpE two-component system of Escherichia coli is still controversial with respect to the nature of the stimulus that is perceived by the sensor kinase KdpD. Limiting potassium concentrations in the medium or high osmolality leads to KdpD/KdpE signal transduction, resulting in kdpFABC expression. It has been hypothesized that changes in turgor are sensed by KdpD through alterations in the physical state of the cytoplasmic membrane. However, in this study the quantitative determination of expression levels of the kdpFABC operon revealed that the system responds very effectively to K(+)-limiting conditions in the medium but barely and to various degrees to salt and sugar stress. Since the current view of stimulus perception calls for mainly intracellular parameters, which might be sensed by KdpD, we set out to test the cytoplasmic concentrations of ATP, K(+), Na(+), glutamate, proline, glycine, trehalose, putrescine, and spermidine under K(+)-limiting conditions. As a first result, the determination of the cytoplasmic volume, which is a prerequisite for such measurements, revealed that a transient shrinkage of the cytoplasmic volume, which is indicative of a reduction in turgor, occurred only under osmotic upshift but not under K(+)-limiting conditions. Furthermore, the intracellular ATP concentration significantly increased under osmotic upshift, whereas only a slight increase occurred after a potassium downshift. Finally, the cytoplasmic K(+) concentration rose severalfold only after an osmotic upshock. For the first time, these data indicate that stimulus perception by KdpD correlates neither with changes in the cytoplasmic volume nor with changes in the intracellular ATP or K(+) concentration or those of the other solutes tested. In conclusion, we propose that a reduction in turgor cannot be the stimulus for KdpD.

K+-translocating KdpFABC P-type ATPase from Escherichia coli acts as a functional and structural dimer.

The membrane-embedded K (+)-translocating KdpFABC complex from Escherichia coli belongs to the superfamily of P-type ATPases, which share common structural features as well as a well-studied catalytic mechanism. However, little is known about the oligomeric state of this class of enzymes. For many P-type ATPases, such as the Na (+)/K (+)-ATPase, Ca (2+)-ATPase, or H (+)-ATPase, an oligomeric state has been shown or is at least discussed but has not yet been characterized in detail. In the KdpFABC complex, kinetic analyses already indicated the presence of two cooperative ATP-binding sides within the functional enzyme and, thus, also point in the direction of a functional oligomer. However, the nature of this oligomeric state has not yet been fully elucidated. In the present work, a close vicinity of two KdpB subunits within the functional KdpFABC complex could be demonstrated by chemical cross-linking of native cysteine residues using copper 1,10-phenanthroline. The cysteines responsible for cross-link formation were identified by mutagenesis. Cross-linked and non-cross-linked KdpFABC complexes eluted with the same apparent molecular weight during gel filtration, which corresponded to the molecular weight of a homodimer, thereby clearly indicating that the KdpFABC complex was purified as a dimer. Isolated KdpFABC complexes were analyzed by transmission electron microscopy and exhibited an approximately 1:1 distribution of mono- and dimeric particles. Finally, reconstituted functional KdpFABC complexes were site-directedly labeled with flourescent dyes, and intermolecular single-molecule FRET analysis was carried out, from which a dissociation constant for a monomer/dimer equilibrium between 30 and 50 nM could be derived.

Three-dimensional structure of the KdpFABC complex of Escherichia coli by electron tomography of two-dimensional crystals.

The KdpFABC complex (Kdp) functions as a K+ pump in Escherichia coli and is a member of the family of P-type ATPases. Unlike other family members, Kdp has a unique oligomeric composition and is notable for segregating K+ transport and ATP hydrolysis onto separate subunits (KdpA and KdpB, respectively). We have produced two-dimensional crystals of the KdpFABC complex within reconstituted lipid bilayers and determined its three-dimensional structure from negatively stained samples using a combination of electron tomography and real-space averaging. The resulting map is at a resolution of 2.4 nm and reveals a dimer of Kdp molecules as the asymmetric unit; however, only the cytoplasmic domains are visible due to the lack of stain penetration within the lipid bilayer. The sizes of these cytoplasmic domains are consistent with Kdp and, using a pseudo-atomic model, we have described the subunit interactions that stabilize the Kdp dimer within the larger crystallographic array. These results illustrate the utility of electron tomography in structure determination of ordered assemblies, especially when disorder is severe enough to hamper conventional crystallographic analysis.

Identity, beer spoiling and biofilm forming potential of yeasts from beer bottling plant associated biofilms.

Wild yeasts were isolated from process surfaces of two breweries. In total, 41 strains were obtained and differentiated by cultivation on CuSO(4) or crystal violet containing selective media, by fatty acid profiling and by a restriction analysis of the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene. The restriction analysis showed the highest differentiating capacity and resulted in eleven groups. These groups were identified by the API ID 32 C kit or by sequencing the D1/D2 region of the 26S rRNA gene. Most of the wild yeasts were identified as Saccharomyces cerevisiae (46% of all isolates) and Candida pelliculosa (anamorph: Pichia anomala) (24%). No obvious differences were detected between the two breweries. While all of the S. cerevisiae isolates were able to grow in beer, only six out of 10 C. pelliculosa strains were able to tolerate this substrate. However, most of the C. pelliculosa strains showed biofilm formation in a microplate assay, but none of the S. cerevisiae isolates. Therefore, it is assumed that the former species is involved in attachment and primary biofilm formation on beer bottling plants, while S. cerevisiae is a late colonizer of a preformed biofilm but increased the beer spoiling potential of the biofilm.

The K+-translocating KdpFABC complex from Escherichia coli: a P-type ATPase with unique features.

The prokaryotic KdpFABC complex from the enterobacterium Escherichia coli represents a unique type of P-type ATPase composed of four different subunits, in which a catalytically active P-type ATPase has evolutionary recruited a potassium channel module in order to facilitate ATP-driven potassium transport into the bacterial cell against steep concentration gradients. This unusual composition entails special features with respect to other P-type ATPases, for example the spatial separation of the sites of ATP hydrolysis and substrate transport on two different polypeptides within this multisubunit enzyme complex, which, in turn, leads to an interesting coupling mechanism. As all other P-type ATPases, also the KdpFABC complex cycles between the so-called E1 and E2 states during catalysis, each of which comprises different structural properties together with different binding affinities for both ATP and the transport substrate. Distinct configurations of this transport cycle have recently been visualized in the working enzyme. All typical features of P-type ATPases are attributed to the KdpB subunit, which also comprises strong structural homologies to other P-type ATPase family members. However, the translocation of the transport substrate, potassium, is mediated by the KdpA subunit, which comprises structural as well as functional homologies to MPM-type potassium channels like KcsA from Streptomyces lividans. Subunit KdpC has long been thought to exhibit an FXYD protein-like function in the regulation of KdpFABC activity. However, our latest results are in favor of the notion that KdpC might act as a catalytical chaperone, which cooperatively interacts with the nucleotide to be hydrolyzed and, thus, increases the rather untypical weak nucleotide binding affinity of the KdpB nucleotide binding domain.

The conserved dipole in transmembrane helix 5 of KdpB in the Escherichia coli KdpFABC P-type ATPase is crucial for coupling and the electrogenic K+-translocation step.

The KdpFABC complex of Escherichia coli, a high-affinity K+-uptake system, belongs to the group of P-type ATPases and is responsible for ATP-driven K+ uptake in the case of K+ limitation. Sequence alignments identified two conserved charged residues, D583 and K586, which are located at the center of transmembrane helix 5 (TM 5) of the catalytic KdpB subunit, and which are supposed to establish a dipole involved in energy coupling. Cells in which the two charges were eliminated or inverted by mutagenesis displayed a clearly slower growth rate with respect to wild-type cells under K+-limiting conditions. Purified KdpFABC complexes from several K586 mutants and a D583K:K586D double mutant showed a reduced K+-stimulated ATPase activity together with an increased resistance to orthovanadate. Upon reconstitution into liposomes, only the conservative K586R mutant was able to facilitate K+ transport, whereas the elimination of the positive charge at position 586 as well as inverting the charges at positions 583 and 586 (D583K:K586D) led to an uncoupling of ATP hydrolysis and K+ transport. Electrophysiological measurements with KdpFABC-containing proteoliposomes adsorbed to planar lipid bilayers revealed that in case of the D583K:K586D double mutant the characteristic K+-independent electrogenic step within the reaction cycle is lacking, thereby clearly arguing for an exact positioning of the dipole for coupling within the functional enzyme complex. In addition, these findings strongly suggest that the dipole residues in KdpB are not directly responsible for the characteristic electrogenic reaction step of KdpFABC, which most likely occurs within the K+-translocating KdpA subunit.

Common patterns and unique features of P-type ATPases: a comparative view on the KdpFABC complex from Escherichia coli (Review).

P-type ATPases are ubiquitously abundant primary ion pumps, which are capable of transporting cations across the cell membrane at the expense of ATP. Since these ions comprise a large variety of vital biochemical functions, nature has developed rather sophisticated transport machineries in all kingdoms of life. Due to the importance of these enzymes, representatives of both eu- and prokaryotic as well as archaeal P-type ATPases have been studied intensively, resulting in detailed structural and functional information on their mode of action. During catalysis, P-type ATPases cycle between the so-called E1 and E2 states, each of which comprising different structural properties together with different binding affinities for both ATP and the transport substrate. Crucial for catalysis is the reversible phosphorylation of a conserved aspartate, which is the main trigger for the conformational changes within the protein. In contrast to the well-studied and closely related eukaryotic P-type ATPases, much less is known about their homologues in bacteria. Whereas in Eukarya there is predominantly only one subunit, which builds up the transport system, in bacteria there are multiple polypeptides involved in the formation of the active enzyme. Such a rather unusual prokaryotic P-type ATPase is the KdpFABC complex of the enterobacterium Escherichia coli, which serves as a highly specific K(+) transporter. A unique feature of this member of P-type ATPases is that catalytic activity and substrate transport are located on two different polypeptides. This review compares generic features of P-type ATPases with the rather unique KdpFABC complex and gives a comprehensive overview of common principles of catalysis as well as of special aspects connected to distinct enzyme functions.

The extension of the fourth transmembrane helix of the sensor kinase KdpD of Escherichia coli is involved in sensing.

The KdpD sensor kinase and the KdpE response regulator control expression of the kdpFABC operon coding for the KdpFABC high-affinity K+ transport system of Escherichia coli. In search of a distinct part of the input domain of KdpD which is solely responsible for K+ sensing, sequences of kdpD encoding the transmembrane region and adjacent N-terminal and C-terminal extensions were subjected to random mutagenesis. Nine KdpD derivatives were identified that had lost tight regulation of kdpFABC expression. They all carried single amino acid replacements located in a region encompassing the fourth transmembrane helix and the adjacent arginine cluster of KdpD. All mutants exhibited high levels of kdpFABC expression regardless of the external K+ concentration. However, 3- to 14-fold induction was observed under extreme K+-limiting conditions and in response to an osmotic upshift when sucrose was used as an osmolyte. These KdpD derivatives were characterized by a reduced phosphatase activity in comparison to the autokinase activity in vitro, which explains constitutive expression. Whereas for wild-type KdpD the autokinase activity and also, in turn, the phosphotransfer activity to KdpE were inhibited by increasing concentrations of K+, both activities were unaffected in the KdpD derivatives. These data clearly show that the extension of the fourth transmembrane helix encompassing the arginine cluster is mainly involved in sensing both K+ limitation and osmotic upshift, which may not be separated mechanistically.

Synthesis, and structural and biological studies of efrapeptin C analogues.

A series of analogues of efrapeptin C (1), with variations in the central tripeptide epitope (positions 6-8), were prepared by a combination of solid- and solution-phase peptide syntheses. The conformations of the modified compounds 2-6 were investigated by circular-dichroism (CD) spectroscopy to differentiate between 3(10)- and alpha-helical secondary structures. The inhibitory activities of the new compounds towards F(1)-ATPase from E. coli were determined. The modified congeners 3-5 were less active by one order of magnitude compared to 1 (K(i) 10 microM), and 6 was completely inactive. Our experiments demonstrate that the flexible, central tripeptide epitope, comprising positions 6-8 in 1, is crucial for molecular recognition, even slight sequence modifications being hardly tolerated.

Channel-mediated potassium uptake in Helicobacter pylori is essential for gastric colonization.

To date, the biological role of prokaryotic K(+) channels remains unknown. Helicobacter pylori contains a gene encoding a putative K(+) channel (HpKchA) of the two-transmembrane RCK (regulation of K(+) conductance) domain family, but lacks known bacterial K(+) uptake systems. A H. pylori DeltahpKchA mutant presented a strong growth defect at low K(+) concentration, which was compensated by KCl addition. The role of the separate RCK domain was investigated in H. pylori by mutagenesis of its internal start codon, which led to a K(+)-dependent intermediate growth phenotype, consistent with RCK activating channel function. Tagging HpKchA C-terminally, we detected a 1:1 stoichiometry of the full-length HpKchA and the separate RCK domain. We constructed single amino-acid exchanges within the unusual selectivity filter of HpKchA (ATGFGA) in H. pylori and observed complete loss (G74A), a slight defect (G76A or F75G) or wild-type (A77D) channel function. HpKchA was essential for colonization of the murine stomach. These data show, for the first time, a biological function for a prokaryotic K(+) channel, as a K(+) uptake system, essential for the persistence of H. pylori in the gastric environment.

Lettuce for human consumption collected in Costa Rica contains complex communities of culturable oxytetracycline- and gentamicin-resistant bacteria.

The present widespread use of antimicrobials in crop farming is based upon their successful application in human medicine. However, recent evidence suggests that the massive anthropogenic release of antimicrobials into the biosphere has selected for resistant bacteria and facilitated the transfer of resistance genes among them. This work deals with the examination of iceberg lettuce collected at 10 farms from two regions in Costa Rica. Farmers from nine sampling sites regularly apply commercial formulations containing gentamicin, oxytetracycline, streptomycin, or a combination of them without being able to indicate how often and how much of these products have been sprayed onto the crops. One organic farm was also investigated for comparative purposes. Oxytetracycline- and gentamicin-resistant bacteria were abundantly detected using selective enrichment cultures. Furthermore, colony mixtures from selective plates were characterized by chemotaxonomical and molecular fingerprinting methods. Both types of resistant communities accounted for a significant fraction of all culturable bacteria and included several resistance genes as well as factors for their potential horizontal transfer. Given the fact that lettuce is eaten raw, it may contribute to the dissemination of antimicrobial-resistant bacteria and/or their resistance genes from the environment to the microbial biota of the human intestine.

ATP binding properties of the soluble part of the KdpC subunit from the Escherichia coli K(+)-transporting KdpFABC P-type ATPase.

P-Type ATPases catalyze the transport of cations across the cell envelope via site-specific hydrolysis of ATP. Modulation of enzyme activity by additional small subunits and/or a second regulatory nucleotide binding site is still a subject of discussion. In the K(+)-transporting KdpFABC complex of Escherichia coli, KdpB resembles the catalytic P-type ATPase subunit, but ATP binding also occurs in the essential but noncatalytic subunit, KdpC. For further characterization, the soluble portion of KdpC (KdpC(sol), residues Asn39-Glu190) was synthesized separately and purified to homogeneity via affinity and size exclusion chromatography. Protein integrity was confirmed by N-terminal sequencing, mass spectrometry, and circular dichroism spectroscopy, which revealed an alpha-helical content of 44% together with an 8% beta-sheet conformation consistent with the values deduced from the primary sequence. The overall protein structure was not affected by the addition of ATP to a concentration of up to 2 mM. In contrast, labeling of KdpC(sol) with the photoreactive ATP analogue 8-azido-ATP resulted in the specific incorporation of one molecule of 8-azido-ATP per peptide. No labeling could be observed upon denaturation of the protein with 0.2% sodium dodecyl sulfate, which suggests the presence of a structured nucleotide binding site. Labeling could be inhibited by preincubation with either ATP, ADP, AMP, GTP, or CTP, thus demonstrating a low specificity for nucleotides. Following 8-azido-ATP labeling and tryptic digestion of KdpC(sol), mass spectrometry showed that ATP binding occurred within the Val144-Lys161 peptide located within the C-terminal part of KdpC, thereby further demonstrating a defined nucleotide binding site. On the basis of these findings, a cooperative model in which the soluble part of KdpC activates catalysis of KdpB is suggested.