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 unusal 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 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 KdpFABC complex from Escherichia coli: a chimeric K+ transporter merging ion pumps with ion channels

The KdpFABC complex represents a multi-subunit ATP-driven potassium pump, which is only found in bacteria and archaea. Based on the properties of the ATP-hydrolyzing subunit (KdpB) the transporter has been classified as a type IA P-type ATPase. However, structural and functional properties of the remaining subunits clearly show homologies to members of the potassium channel as well as the ABC transporter family, thus rendering the KdpFABC complex to represent an inimitable chimera of ion pumps and ion channels. Accordingly, this striking juxtaposition entails special features of KdpFABC with respect to typical members of each of the transporter families, involving not only the concepts but also the structures of ion channels and ion pumps. For example, the sites of ATP hydrolysis and substrate transport are spatially separated on two different polypeptides, which, in turn, leads to a unique coupling mechanism. During catalysis, the KdpFABC complex cycles between two main conformational states, each of which comprises different structural properties together with different binding affinities for both ATP and the transport substrate. These structural configurations have recently been directly visualized in the working enzyme. Translocation of potassium is mediated by the KdpA subunit, which comprises structural as well as functional homologies to potassium channels of the MPM-type. The KdpC subunit participates in the binding of ATP, thus acting as a catalytic chaperone, which increases the ATP binding affinity of the KdpB subunit via a mechanism typical of nucleotide binding in ABC transporters.     

Solution structure of the KdpFABC P-type ATPase from Escherichia coli by electron microscopic single particle analysis

The K⁺-translocating KdpFABC complex from Escherichia coli functions as a high affinity potassium uptake system and belongs to the superfamily of P-type ATPases, although it exhibits some unique features. It comprises four subunits, and the sites of ATP hydrolysis and substrate transport are located on two different polypeptides. No structural data are so far available for elucidating the correspondingly unique mechanism of coupling ion transport and catalysis in this P-type ATPase. By use of electron microscopy and single particle analysis of negatively stained, solubilized KdpFABC complexes, we solved the structure of the complex at a resolution of 19 Å, which allowed us to model the arrangement of subunits within the holoenzyme and, thus, to identify the interfaces between subunits. The model showed that the K⁺-translocating KdpA subunit is in close contact with the transmembrane region of the ATP-hydrolyzing subunit KdpB. The cytosolic C-terminal domain of the KdpC subunit, which is assumed to play a role in cooperative ATP binding together with KdpB, is located in close vicinity to the nucleotide binding domain of KdpB. Overall, the arrangement of subunits agrees with biochemical data and the predictions on subunit interactions.     

The KdpC subunit of the Escherichia coli K+-transporting KdpB P-type ATPase acts as a catalytic chaperone

In Bacteria and Archaea, high-affinity potassium uptake is mediated by the ATP-driven KdpFABC complex. On the basis of the biochemical properties of the ATP-hydrolyzing subunit KdpB, the transport complex is classified as type IA P-type ATPase. However, the KdpA subunit, which promotes K(+) transport, clearly resembles a potassium channel, such that the KdpFABC complex represents a chimera of ion pumps and ion channels. In the present study, we demonstrate that the blending of these two groups of transporters in KdpFABC also entails a nucleotide-binding mechanism in which the KdpC subunit acts as a catalytic chaperone. This mechanism is found neither in P-type ATPases nor in ion channels, although parallels are found in ABC transporters. In the latter, the ATP nucleotide is coordinated by the LSGGQ signature motif via double hydrogen bonds at a conserved glutamine residue, which is also present in KdpC. High-affinity nucleotide binding to the KdpFABC complex was dependent on the presence of this conserved glutamine residue in KdpC. In addition, both ATP binding to KdpC and ATP hydrolysis activity of KdpFABC were sensitive to the accessibility, presence or absence of the hydroxyl groups at the ribose moiety of the nucleotide. Furthermore, the KdpC subunit was shown to interact with the nucleotide-binding loop of KdpB in an ATP-dependent manner around the ATP-binding pocket, thereby increasing the ATP-binding affinity by the formation of a transient KdpB/KdpC/ATP ternary complex.     

FITC binding site and p-nitrophenyl phosphatase activity of the Kdp-ATPase of Escherichia coli

The KdpFABC complex of Escherichia coli, which belongs to the P-type ATPase family, has a unique structure, since catalytic activity (KdpB) and the capacity to transport potassium ions (KdpA) are located on different subunits. We found that fluorescein 5-isothiocyanate (FITC) inhibits ATPase activity, probably by covalently modifying lysine 395 in KdpB. In addition, we observed that the KdpFABC complex is able to hydrolyze p-nitrophenyl phosphate (pNPP) in a Mg(2+)-dependent reaction. The pNPPase activity is inhibited by FITC and o-vanadate. Low concentrations of ATP (1-30 microM) stimulate the pNPPase activity, while concentrations of >500 microM are inhibitory. This behavior can be explained either by a regulatory ATP binding site, where ATP hydrolysis is required, or by proposing an interactive dimer. The notion that FITC inhibits pNPPase and ATPase activity supports the idea that the catalytic domain of KdpB is much more compact than other P-type ATPases, like Na(+),K(+)-ATPase, H(+),K(+)-ATPase, and Ca(2+)-ATPase.     

An Intracellular Pathway Controlled by the N-terminus of the Pump Subunit Inhibits the Bacterial KdpFABC Ion Pump in High K+ Conditions

The heterotetrameric bacterial KdpFABC transmembrane protein complex is an ion channel-pump hybrid that consumes ATP to import K⁺ against its transmembrane chemical potential gradient in low external K⁺ environments. The KdpB ion-pump subunit of KdpFABC is a P-type ATPase, and catalyses ATP hydrolysis. Under high external K⁺ conditions, K⁺ can diffuse into the cells through passive ion channels. KdpFABC must therefore be inhibited in high K⁺ conditions to conserve cellular ATP. Inhibition is thought to occur via unusual phosphorylation of residue Ser162 of the TGES motif of the cytoplasmic A domain. It is proposed that phosphorylation most likely traps KdpB in an inactive E1-P like conformation, but the molecular mechanism of phosphorylation-mediated inhibition remains unknown. Here, we employ molecular dynamics (MD) simulations of the dephosphorylated and phosphorylated versions of KdpFABC to demonstrate that phosphorylated KdpB is trapped in a conformation where the ion-binding site is hydrated by an intracellular pathway between transmembrane helices M1 and M2 which opens in response to the rearrangement of cytoplasmic domains resulting from phosphorylation. Cytoplasmic access of water to the ion-binding site is accompanied by a remarkable loss of secondary structure of the KdpB N-terminus and disruption of a key salt bridge between Glu87 in the A domain and Arg212 in the P domain. Our results provide the molecular basis of a unique mechanism of regulation amongst P-type ATPases, and suggest that the N-terminus has a significant role to play in the conformational cycle and regulation of KdpFABC.     

Plant P-type ATPases

P-type ATPases are a superfamily of membrane proteins involved in many physiological processes that are fundamental for all living organisms. Using ATP, they can transport a variety of ions and other substances across all types of cell membranes against a concentration electrochemical gradient. P-type ATPases form a phosphorylated intermediate and are sensitive to vanadate. Based on evolutionary relations and sequence homology, P-type ATPases are divided into five major families. All P-type ATPases share a simple structure and mechanism, but also possess domains characteristic for each family, which are crucial for substrate specificity. These proteins usually have a single subunit with eight to twelve transmembrane segments, a large central cytoplasmic domain with the conservative ATP binding site along with N and C termini exposed to the cytoplasm. Because of variety of proteins that belong to P-type ATPase superfamily, in this review the comparison of functional and structure properties of plant cells P-type ATPases is presented, as well as their important role in adaptation to environmental stress.     

Prokaryotic Kdp-ATPase: recent insights into the structure and function of KdpB

P-type ATPases are amongst the most abundant enzymes that are responsible for active transport of ions across biological membranes. Within the last 5 years a detailed picture of the structure and function of these transport ATPases has emerged. Here, we report on the recent progress in elucidating the molecular mechanism of a unique, prokaryotic member of P-type ATPases, the Kdp-ATPase. The review focuses on the catalytic parts of the central subunit, KdpB. The structure of the nucleotide-binding domain was solved by NMR spectroscopy at high resolution and a model of the nucleotide-binding mode was presented. The nucleotide turned out to be 'clipped' into the binding pocket by a pi-pi interaction to F377 on one side and a cation-pi interaction to K395 on the other. The 395KGXXD/E motif and thus the nucleotide-binding mode seems to be conserved in all P-type ATPases, except the heavy metal-transporting (class IB) ATPases. Hence, it can be concluded that KdpB is currently misgrouped as class IA. Mutational studies on two highly conserved residues (D583 and K586) in the transmembrane helix 5 of KdpB revealed that they are indispensable in coupling ATP hydrolysis to ion translocation. Based on these results, two possible pathways for the reaction cycle are discussed.     

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.     

Abolishment of proton pumping and accumulation in the E1P conformational state of a plant plasma membrane H+-ATPase by substitution of a conserved aspartyl residue in transmembrane segment 6

The plasma membrane H(+)-ATPase AHA2 of Arabidopsis thaliana, which belongs to the P-type ATPase superfamily of cation-transporting ATPases, pumps protons out of the cell. To investigate the mechanism of ion transport by P-type ATPases we have mutagenized Asp(684), a residue in transmembrane segment M6 of AHA2 that is conserved in Ca(2+)-, Na(+)/K(+)-, H(+)/K(+)-, and H(+)-ATPases and which coordinates Ca(2+) ions in the SERCA1 Ca(2+)-ATPase. We describe the expression, purification, and biochemical analysis of the Asp(684) --> Asn mutant, and provide evidence that Asp(684) in the plasma membrane H(+)-ATPase is required for any coupling between ATP hydrolysis, enzyme conformational changes, and H(+)-transport. Proton pumping by the reconstituted mutant enzyme was completely abolished, whereas ATP was still hydrolyzed. The mutant was insensitive to the inhibitor vanadate, which preferentially binds to P-type ATPases in the E(2) conformation. During catalysis the Asp(684) --> Asn enzyme accumulated a phosphorylated intermediate whose stability was sensitive to addition of ADP. We conclude that the mutant enzyme is locked in the E(1) conformation and is unable to proceed through the E(1)P-E(2)P transition.     

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.     

Single amino acid substitution in the putative transmembrane helix V in KdpB of the KdpFABC complex of Escherichia coli uncouples ATPase activity and ion transport

The KdpFABC complex, found in a variety of prokaryotes, is an emergency potassium uptake system which belongs to the family of P-type ATPases. Site-directed mutagenesis of the charged residues aspartate 583 and lysine 586 in the putative transmembrane helix V of subunit KdpB revealed that these charges are involved in the coupling of ATP hydrolysis and ion translocation. Phenotypic characterization of KdpFABC derivatives carrying alterations at either D583 or K586 demonstrated that only restoration of charges at these positions allowed growth on low potassium concentrations. Substitutions, which eliminated the negative charge at position 583, did not allow growth below 15 mM potassium on solid media. In contrast, substitutions of the positive charge at position 586 allowed growth down to 0.3 mM potassium. Purified KdpFABC complexes carrying these substitutions exhibited ATPase activity, which was, however, found to be comparatively resistant to o-vanadate. Furthermore, elimination of the charges led to a complete loss of ion-stimulated ATPase activity, though the rate of hydrolysis was comparable to wild-type activity, indicating an uncoupling between ATP hydrolysis and ion translocation. This fact was substantiated by reconstitution experiments, in which the D583A complex was unable to facilitate ion translocation, whereas the D583E mutant complex still exhibited such activity. On the basis of these results, a new transport model for the Kdp-ATPase is presented here, in which the amino acids D583 and K586 are supposed to play a role in the gating mechanism of the complex. Furthermore, movement of the charged side chains could have a direct influence on the free energy profile within the potassium transporting subunit KdpA, thereby facilitating ion transport against the concentration gradient into the cytosol.     

Orphan enzyme or patriarch of a new tribe: the arsenic resistance ATPase of bacterial plasmids

The plasmid-determined arsenite and antimonite efflux ATPase of bacteria differs from other membrane transport ATPases, which are classified into several families (such as the F₀F₁-type H⁺-translocating ATP synthases, the related vacuolar H⁺-translocating ATPases, the P-type cation-translocating ATPases, and the superfamily which includes the periplasmic binding-protein-dependent systems in Gram-negative bacteria, the human multidrug resistance P-glycoprotein, and the cystic fibrosis transport regulator). The amino acid sequences of the components of the arsenic resistance system are not similar to known ATPase proteins. New findings with the arsenic resistance operons of bacterial plasmids suggest that instead of being an orphan the Ars system will now be the first recognized member of a new class of ATPases. Furthermore, fundamental questions of energy-coupling (ATP-driven or chemiosmotic) have recently been raised and the finding that the arsC gene product is a soluble enzyme that reduces arsenate to arsenite changes the previous picture of the functioning of this widespread bacterial system.     

The p-type ATPase superfamily

P-type ATPases function to provide homeostasis in higher eukaryotes, but they are essentially ubiquitous, being found in all domains of life. Thever and Saier [J Memb Biol 2009;229:115-130] recently reported analyses of eukaryotic P-type ATPases, dividing them into nine functionally characterized and 13 functionally uncharacterized (FUPA) families. In this report, we analyze P-type ATPases in all major prokaryotic phyla for which complete genome sequence data are available, and we compare the results with those for eukaryotic P-type ATPases. Topological type I (heavy metal) P-type ATPases predominate in prokaryotes (approx. tenfold) while type II ATPases (specific for Na(+),K(+), H(+) Ca(2+), Mg(2+) and phospholipids) predominate in eukaryotes (approx. twofold). Many P-type ATPase families are found exclusively in prokaryotes (e.g. Kdp-type K(+) uptake ATPases (type III) and all ten prokaryotic FUPA familes), while others are restricted to eukaryotes (e.g. phospholipid flippases and all 13 eukaryotic FUPA families). Horizontal gene transfer has occurred frequently among bacteria and archaea, which have similar distributions of these enzymes, but rarely between most eukaryotic kingdoms, and even more rarely between eukaryotes and prokaryotes. In some bacterial phyla (e.g. Bacteroidetes, Flavobacteria and Fusobacteria), ATPase gene gain and loss as well as horizontal transfer occurred seldom in contrast to most other bacterial phyla. Some families (i.e. Kdp-type ATPases) underwent far less horizontal gene transfer than other prokaryotic families, possibly due to their multisubunit characteristics. Functional motifs are better conserved across family lines than across organismal lines, and these motifs can be family specific, facilitating functional predictions. In some cases, gene fusion events created P-type ATPases covalently linked to regulatory catalytic enzymes. In one family (FUPA Family 24), a type I ATPase gene (N-terminal) is fused to a type II ATPase gene (C-terminal) with retention of function only for the latter. Several pseudogene-encoded nonfunctional ATPases were identified. Genome minimalization led to preferential loss of P-type ATPase genes. We suggest that in prokaryotes and some unicellular eukaryotes, the primary function of P-type ATPases is protection from extreme environmental stress conditions. The classification of P-type ATPases of unknown function into phylogenetic families provides guides for future molecular biological studies.     

ATP dependent charge movement in ATP7B Cu+-ATPase is demonstrated by pre-steady state electrical measurements

ATP7B is a copper dependent P-type ATPase, required for copper homeostasis. Taking advantage of high yield heterologous expression of recombinant protein, we investigated charge transfer in ATP7B. We detected charge displacement within a single catalytic cycle upon ATP addition and formation of phosphoenzyme intermediate. We attribute this charge displacement to movement of bound copper within ATP7B. Based on specific mutations, we demonstrate that enzyme activation by copper requires occupancy of a site in the N-terminus extension which is not present in other transport ATPases, as well as of a transmembrane site corresponding to the cation binding site of other ATPases.     

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.     

Inter-domain motions of the N-domain of the KdpFABC complex, a P-type ATPase, are not driven by ATP-induced conformational changes

P-type ATPases are involved in the active transport of ions across biological membranes. The KdpFABC complex (P-type ATPase) of Escherichia coli is a high-affinity K+ uptake system that operates only when the cell experiences osmotic stress or K+ limitation. Here, we present the solution structure of the nucleotide binding domain of KdpB (backbone RMSD 0.17 A) and a model of the AMP-PNP binding mode based on intermolecular distance restraints. The calculated AMP-PNP binding mode shows the purine ring of the nucleotide to be "clipped" into the binding pocket via a pi-pi-interaction to F377 on one side and a cation-pi-interaction to K395 on the other. This binding mechanism seems to be conserved in all P-type ATPases, except the heavy metal transporting ATPases (type IB). Thus, we conclude that the Kdp-ATPase (currently type IA) is misgrouped and has more similarities to type III ATPases. The KdpB N-domain is the smallest and simplest known for a P-type ATPase, and represents a minimal example of this functional unit. No evidence of significant conformational changes was observed within the N-domain upon nucleotide binding, thus ruling out a role for ATP-induced conformational changes in the reaction cycle.