Molecular mechanism of the P-type ATPases

The recent determination of the structure of the Ca²⁺-ATPase of sarcoplasmic reticulum to atomic resolution in the Ca²⁺-bound state and to near atomic resolution in the Ca²⁺-free, decavanadate-bound state has paved the way for an ultimate complete understanding of the molecular mechanism of the P-type ATPases. Analysis of this new structure information together with the large amount of biochemical information about these enzymes that preceded it has produced important new revelations about how the P-type ATPases work. Most models propose that these transporters operate by a strictly conformational energy coupling mechanism in which conformational changes in the large cytoplasmic head region mechanically drive the ions to be transported from their binding sites in the transmembrane helix region 50 Å away. However, while these enzymes do indeed undergo profound conformational changes, the available evidence suggests that they do not mechanically transduce the chemical energy of ATP hydrolysis into transmembrane ion gradients via these conformational changes. As an alternative, it is proposed that the effects of the chemical events that occur at the phosphorylation/dephosphorylation site in the cytoplasmic region are exerted on the ion-binding sites via two well-defined charge transfer pathways that electronically connect the chemical reaction site with the site of ion binding. The recognition of these charge transfer pathways provides rational explanations of all of the key biochemical features of the P-type ATPase catalytic cycle. Thus, although a few details await elucidation, a nearly complete understanding of the P-type ATPase reaction mechanism may be at hand.     

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.     

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.     

Conserved Asp684 in transmembrane segment M6 of the plant plasma membrane P-type proton pump AHA2 is a molecular determinant of proton translocation

The mechanism of proton pumping by P-type H(+)-ATPases is still unclear. In the plant P-type plasma membrane H(+)-ATPase AHA2, two charged residues, Arg(655) and Asp(684), are conserved in transmembrane segments M5 and M6, respectively, a region that has been shown be contribute to ion coordination in related P-type ATPases. Substitution of Arg(655) with either alanine or aspartate resulted in mutant enzymes exhibiting a significant shift in the P-type ATPase E(1)P-E(2)P conformational equilibrium. The mutant proteins accumulated in the E(1)P conformation, but were capable of conducting proton transport. This points to an important role of Arg(655) in the E(1)P-E(2)P conformational transition. The presence of a carboxylate moiety at position Asp(684) proved essential for coupling between initial proton binding and proton pumping. The finding that the carboxylate side chain of Asp(684) contributes to the proton-binding site and appears to function as an absolutely essential proton acceptor along the proton transport pathway is discussed in the context of a possible proton pumping mechanism of P-type H(+)-ATPases.     

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.     

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.     

Structures of P-type transporting ATPases and chromosomal locations of their genes

P-type ATPases (E1E2-ATPases) are primary active transporters which form phospho-intermediates during their catalytic cycle. They are classified into P1 to P4 based on the primary structure and potential transmembrane segments. Although the classic P-type ATPases are cation transporters, two new members have recently been found; one is a flippase catalyzing the flip-flop movement of aminophospholipids, but the substrate and function of the other one remain unknown. It would be interesting to determine whether the cations and aminophospholipids are transported by similar or different mechanisms. P-type ATPases are believed to have been derived from a common ancestor, and their genes are found to be distributed in various chromosomal loci. However, gene duplication events can be traced from the tandem arrangement of genes and their linkage map. Na+/K+- and H+/K+-ATPases have not only closely related a subunits but also similar beta subunits. Renal Na+/K+-ATPase has an additional subunit gamma. Similar small polypeptides (phospholemman, Mat-8 and CHIF), which induce Cl- and K+ currents, have been found. The idea of their functional and structural coupling with P-type ATPases, especially with H+/K+-ATPase, is intriguing. Each P-type ATPase must have specific domains or sequences for its intracellular trafficking (sorting, retention and recycling). Identification of such regions and studies on the molecules playing role in their recognition may facilitate the unveiling of various cellular processes regulated by P-type ATPases.     

ATP-binding to P-type ATPases as revealed by biochemical, spectroscopic, and crystallographic experiments

P-type ATPases form a large family of cation translocating ATPases. Recent progress in crystallography yielded several high-resolution structures of Ca(2+)-ATPase from sarco(endo)plasmic reticulum (SERCA) in various conformations. They could elucidate the conformational changes of the enzyme, which are necessary for the translocation of cations, or the mechanism that explains how the nucleotide binding is coupled to the cation transport. However, crystals of proteins are usually obtained only under conditions that significantly differ from the physiological ones and with ligands that are incompatible with the enzyme function, and both of these factors can inevitably influence the enzyme structure. Biochemical (such as mutagenesis, cleavage, and labeling) or spectroscopic experiments can yield only limited structural information, but this information could be considered relevant, because measurement can be performed under physiological conditions and with true ligands. However, interpretation of some biochemical or spectroscopic data could be difficult without precise knowledge of the structure. Thus, only a combination of both these approaches can extract the relevant information and identify artifacts. Briefly, there is good agreement between crystallographic and other experimental data concerning the overall shape of the molecule and the movement of cytoplasmic domains. On the contrary, the E1-AMPPCP crystallographic structure is, in details, in severe conflict with numerous spectroscopic experiments and probably does not represent the physiological state. Notably, the E1-ADP-AlF(4) structure is almost identical to the E1-AMPPCP, again suggesting that the structure is primarily determined by the crystal-growth conditions. The physiological relevance of the E2 and E2-P structures is also questionable, because the crystals were prepared in the presence of thapsigargin, which is known to be a very efficient inhibitor of SERCA. Thus, probably only crystals of E1-2Ca conformation could reflect some physiological state. Combination of biochemical, spectroscopic, and crystallographic data revealed amino acids that are responsible for the interaction with the nucleotide. High sequence homology of the P-type ATPases in the cytoplasmic domains enables prediction of the ATP-interacting amino acids also for other P-type ATPases.     

Diving Into the Lipid Bilayer to Investigate the Transmembrane Organization and Conformational State Transitions of P-type Ion ATPases

Although membrane proteins constitute more than 20% of the total proteins, the structures of only a few are known in detail. An important group of integral membrane proteins are ion-transporting ATPases of the P-type family, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. There are several crystal structures of the sarcoplasmic reticulum Ca(2+) pump (SERCA) revealing different conformations, and recently, crystal structures of the H(+)-ATPase and the Na(+)/K(+)-ATPase were reported as well. However, there are no atomic resolution structures for other P-type ATPases including the plasma membrane calcium pump (PMCA), which is integral to cellular Ca(2+) signaling. Crystallization of these proteins is challenging because there is often no natural source from which the protein can be obtained in large quantities, and the presence of multiple isoforms in the same tissue further complicates efforts to obtain homogeneous samples suitable for crystallization. Alternative techniques to study structural aspects and conformational transitions in the PMCAs (and other P-type ATPases) have therefore been developed. Specifically, information about the structure and assembly of the transmembrane domain of an integral membrane protein can be obtained from an analysis of the lipid-protein interactions. Here, we review recent efforts using different hydrophobic photo-labeling methods to study the non-covalent interactions between the PMCA and surrounding phospholipids under different experimental conditions, and discuss how the use of these lipid probes can reveal valuable information on the membrane organization and conformational state transitions in the PMCA, Na(+)/K(+)-ATPase, and other P-type ATPases.     

Characterization of Na,K-ATPase and H,K-ATPase enzymes with glycosylation-deficient beta-subunit variants by voltage-clamp fluorometry in Xenopus oocytes

The role of N-linked glycosylation of beta-subunits in the functional properties of the oligomeric P-type ATPases Na,K- and H,K-ATPase has been examined by expressing glycosylation-deficient Asn-to-Gln beta-variants in Xenopus oocytes. For both ATPases, the absence of the huge N-linked oligosaccharide moiety on the beta-subunit does not affect alpha/beta coassembly, plasma membrane delivery or functional activity of the holoenzyme. Whereas this is in line with several previous glycosylation studies on Na,K-ATPase, this is the first report showing that the cell surface delivery and enzymatic activity of the gastric H,K-ATPase is unaffected by the lack of N-linked glycosylation. Sulfhydryl-specific labeling of introduced cysteine reporter sites with the environmentally sensitive fluorophore tetramethylrhodamine-6-maleimide (TMRM) upon expression in Xenopus oocytes enabled us to further investigate potential effects of the N-glycans on more subtle enzymatic properties, like the distribution between E 1P/E 2P states of the catalytic cycle and the kinetics of the E 1P/E 2P conformational transition under presteady state conditions. For both Na,K-ATPase and H,K-ATPase, we observed differences in neither the voltage-dependent E 1P/E 2P ratio nor the kinetics of the E 1P/E 2P transition between holoenzymes comprising glycosylated and glycosylation-deficient beta-subunits. We conclude that the N-linked glycans on these essential accessory subunits of oligomeric P-type ATPases are dispensable for proper folding, membrane stabilization of the alpha-subunit and transport function itself. Glycosylation is rather important for other cellular functions not relevant in the oocyte expression system, such as intercellular interactions or basolateral versus apical targeting in polarized cells, as demonstrated in other expression systems.     

The conformation of H,K-ATPase determines the nucleoside triphosphate (NTP) selectivity for active proton transport

The gastric H,K-ATPase is related to other cation transport ATPases, for example, Na,K-ATPase and Ca-ATPase, which are called E1-E2 ATPases in recognition of conformational transitions during their respective transport and catalytic cycles. Generally, these ATPases cannot utilize NTPs other than ATP for net ion transport activity. For example, under standard assay conditions, rates of NTP hydrolysis and H+ pumping by the H,K-ATPase for CTP are about 10% of those for ATP and undetectable with GTP, ITP, and UTP. However, we observed that H,K-ATPase will catalyze NTP/ADP phosphate exchange at similar rates for all of these NTPs, suggesting that a common phosphoenzyme intermediate is formed. The present study was undertaken to evaluate the specificity of nucleotides to power the H,K-ATPase and several of its partial reactions, including NTP/ADP exchange, K+-catalyzed phosphatase activity, and proton pumping. Results demonstrate that under conditions that promote the conformational change of the K+ bound form of the enzyme, K.E2, to E1, all NTPs tested support K+-stimulated NTPase activity and H+ pumping up to 30-50% of that with ATP. These conditions include (1) the presence of ADP as well as the NTP energy source and (2) reduced K+ concentration on the cytoplasmic side to approximately 0. These data conform to structural models for E1-E2 ATPases whereby adenosine binding promotes the K.E2 to E1 conformational change and K+ deocclusion.     

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.     

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.     

Ion transport and energy transduction of P-type ATPases: Implications from electrostatic calculations

This paper summarizes our present electrostatic calculations on P-type ATPases and their contribution to understand the molecular details of the reaction mechanisms. One focus was set on analyzing the proton countertransport of the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA1a). Protonation of acidic residues was calculated in dependence of pH for different enzyme states in the reaction cycle of the Ca²⁺-ATPase. We proposed that the acidic Ca²⁺ ligands Glu 771, Asp 800 and Glu 908 participate in the proton countertransport whereas Glu 309 is more likely to serve as a proton shuttle between binding site I and the cytoplasm. Complementary to infrared measurements, we assigned infrared bands to specific Ca²⁺ ligands that are hydrogen bonded. Ion pathways were proposed based on the calculations and structural data. Another focus was set on analyzing the energy transduction mechanism of P-type ATPases. In accordance to electrophysiological experiments, we simulated an electric field across the membrane. The impact of the electric field was studied by an accumulated number of residue conformational and ionization changes on specific transmembrane helices. Our calculations on the Ca²⁺-ATPase and the Na⁺/K⁺-ATPase indicated that the highly conserved transmembrane helix M5 is one structural element that is likely to act as energy transduction element in P-type ATPases. Perspectives and limitations of the electrostatic calculations for future computational studies are pointed out.     

Structural Basis of Substrate-Independent Phosphorylation in a P4-ATPase Lipid Flippase

P4-ATPases define a eukaryotic subfamily of the P-type ATPases, and are responsible for the transverse flip of specific lipids from the extracellular or luminal leaflet to the cytosolic leaflet of cell membranes. The enzymatic cycle of P-type ATPases is divided into autophosphorylation and dephosphorylation half-reactions. Unlike most other P-type ATPases, P4-ATPases transport their substrate during dephosphorylation only, i.e. the phosphorylation half-reaction is not associated with transport. To study the structural basis of the distinct mechanisms of P4-ATPases, we have determined cryo-EM structures of Drs2p-Cdc50p from Saccharomyces cerevisiae covering multiple intermediates of the cycle. We identify several structural motifs specific to Drs2p and P4-ATPases in general that decrease movements and flexibility of domains as compared to other P-type ATPases such as Na⁺/K⁺-ATPase or Ca²⁺-ATPase. These motifs include the linkers that connect the transmembrane region to the actuator (A) domain, which is responsible for dephosphorylation. Additionally, mutation of Tyr380, which interacts with conserved Asp340 of the distinct DGET dephosphorylation loop of P4-ATPases, highlights a functional role of these P4-ATPase specific motifs in the A-domain. Finally, the transmembrane (TM) domain, responsible for transport, also undergoes less extensive conformational changes, which is ensured both by a longer segment connecting TM helix 4 with the phosphorylation site, and possible stabilization by the auxiliary subunit Cdc50p. Collectively these adaptions in P4-ATPases are responsible for phosphorylation becoming transport-independent.     

Mechanism of allosteric effects of ATP on the kinetics of P-type ATPases

The roles of allosteric effects of ATP and protein oligomerisation in the mechanisms of P-type ATPases belong to the most controversial and least well understood topics in the field. Recent crystal structural and kinetic data, however, now allow certain hypotheses to be definitely excluded and consistent hypotheses to be developed. The aim of this review is to critically discuss recent results and, in the light of them, to present a set of conclusions which could form the basis of future research. The major conclusions are: (1) at saturating ATP concentrations P-type ATPases function as monomeric enzymes, (2) the catalytic units of P-type ATPases only possess a single ATP binding site, (3) at non-saturating ATP concentrations P-type ATPases exist as diprotomeric (or higher oligomeric) complexes, (4) protein–protein interactions within a diprotomeric complex enhances the enzymes’ ATP binding affinity, (5) ATP binding to both protomers within a diprotomeric complex causes it to dissociate into two separate monomers. The physiological role of protein–protein interactions within a diprotomer may be to enhance ATP binding affinity so as to scavenge ATP and maximize the ion pumping rate under hypoxic or anoxic conditions. For the first time a structural basis for the well-known ATP allosteric acceleration of the E2 → E1 transition is presented. This is considered to be due to a minimization of steric hindrance between neighbouring protomers because of the ability of ATP to induce a compact conformation of the enzymes’ cytoplasmic domains.     

Proton and calcium pumping P-type ATPases and their regulation of plant responses to the environment

Plant plasma membrane H⁺-ATPases and Ca²⁺-ATPases maintain low cytoplasmic concentrations of H⁺ and Ca²⁺, respectively, and are essential for plant growth and development. These low concentrations allow plasma membrane H⁺-ATPases to function as electrogenic voltage stats, and Ca²⁺-ATPases as “off” mechanisms in Ca²⁺-based signal transduction. Although these pumps are autoregulated by cytoplasmic concentrations of H⁺ and Ca²⁺, respectively, they are also subject to exquisite regulation in response to biotic and abiotic events in the environment. A common paradigm for both types of pumps is the presence of terminal regulatory (R) domains that function as autoinhibitors that can be neutralized by multiple means, including phosphorylation. A picture is emerging in which some of the phosphosites in these R domains appear to be highly, nearly constantly phosphorylated, whereas others seem to be subject to dynamic phosphorylation. Thus, some sites might function as major switches, whereas others might simply reduce activity. Here, we provide an overview of the relevant transport systems and discuss recent advances that address their relation to external stimuli and physiological adaptations.

The regulation of plasma membrane H^+-ATPases and autoinhibited Ca2^+-ATPases exhibits a complex and dynamic network of posttranslational regulation. The regulation of plasma membrane H⁺-ATPases and autoinhibited Ca²⁺-ATPases exhibits a complex and dynamic network of posttranslational regulation.

P-type ATPases are found in all domains of life and constitute a large superfamily of membrane-bound pumps that share a common machinery, including a reaction cycle that involves catalytic phosphorylation of an Asp, resulting in a phosphorylated intermediate (reviewed in Palmgren and Nissen, 2011; (hence the name P-type; Box 1). The catalytic phosphoryl-aspartate intermediate is not to be confused with regulatory phosphorylation, which occurs on Ser, Thr, and Tyr residues. Five major families of P-type ATPases have been characterized (P1–5), each of which is divided into a number of subfamilies (named with letters). Plasma membrane H⁺-ATPases are classified as P3A ATPases, whereas Ca²⁺ pumps constitute P2A and P2B ATPases. In plants, these pumps are best characterized in the model plant Arabidopsis thaliana (Arabidopsis).Box 1Enzymology of P-type ATPases.P-type ATPases (reviewed in Palmgren and Nissen, 2011) alternate between two extreme conformations during their catalytic cycle: a high-affinity (with respect to ATP and the ion to be exported) Enzyme1 (E1) state, and a low-affinity Enzyme2 (E2) state. Many P-type ATPases are autoinhibited by built-in molecular constraints, namely their C- and N-terminal (for plasma membrane H⁺-ATPases; Palmgren et al., 1999) or N-terminal (for P2B Ca²⁺-ATPases; Malmström et al., 1997) regulatory (R) domains of approximately 100 amino acid residues, which act as brakes by stabilizing the pumps in a low-affinity conformation (Palmgren and Nissen, 2011), most likely E2. Neutralizing the R domain results in a shift in conformational equilibrium towards a high-affinity state, likely E1. In this way, the R domains of plasma membrane H⁺-ATPases and Ca²⁺-ATPases allow posttranslational modification events to control the turnover numbers of these pumps. A structure of a plasma membrane H⁺-ATPase (from the distantly related yeast S. cerevisiae) in its autoinhibited state has been solved (Heit et al., 2021). Its R domain is situated adjacent to the P domain, which would suggest that the R domain functions to restrict the conformational flexibility of the pump. Normally, the hydrolysis of ATP and transport are tightly coupled in P-type ATPases. Therefore, P-type ATPases hydrolyze bound ATP as soon as their ligand-binding site(s) in the membrane region are occupied, but not before. Thus, increasing the ligand affinity of an ATPase simultaneously increases its turnover number, provided that the concentration of ATP is not limiting, which is rarely the case in cells. A specific feature of plasma membrane H⁺-ATPases is that in the autoinhibited state, ATP hydrolysis is only loosely coupled to H⁺ pumping, whereas pump activation results in tight coupling, with one H⁺ pumped per ATP split (Pedersen et al., 2018).In response to internal and/or external cues, plasma membrane H⁺-ATPase and Ca²⁺-ATPase activities are controlled by intracellular concentrations of H⁺ and Ca²⁺, respectively, via interacting proteins, through posttranslational modification by phosphorylation, and by regulated trafficking of the pump to and from the plasma membrane. Their regulation sometimes involves changes in gene expression and turnover, although this is rare, perhaps because both processes are time- and energy-consuming (Haruta et al., 2018).     

Metal Fluoride Inhibition of a P-type H+ Pump: STABILIZATION OF THE PHOSPHOENZYME INTERMEDIATE CONTRIBUTES TO POST-TRANSLATIONAL PUMP ACTIVATION*

The plasma membrane H⁺-ATPase is a P-type ATPase responsible for establishing electrochemical gradients across the plasma membrane in fungi and plants. This essential proton pump exists in two activity states: an autoinhibited basal state with a low turnover rate and a low H⁺/ATP coupling ratio and an activated state in which ATP hydrolysis is tightly coupled to proton transport. Here we characterize metal fluorides as inhibitors of the fungal enzyme in both states. In contrast to findings for other P-type ATPases, inhibition of the plasma membrane H⁺-ATPase by metal fluorides was partly reversible, and the stability of the inhibition varied with the activation state. Thus, the stability of the ATPase inhibitor complex decreased significantly when the pump transitioned from the activated to the basal state, particularly when using beryllium fluoride, which mimics the bound phosphate in the E2P conformational state. Taken together, our results indicate that the phosphate bond of the phosphoenzyme intermediate of H⁺-ATPases is labile in the basal state, which may provide an explanation for the low H⁺/ATP coupling ratio of these pumps in the basal state.