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.     

The Structural Basis for Phospholamban Inhibition of the Calcium Pump in Sarcoplasmic Reticulum

P-type ATPases are a large family of enzymes that actively transport ions across biological membranes by interconverting between high (E1) and low (E2) ion-affinity states; these transmembrane transporters carry out critical processes in nearly all forms of life. In striated muscle, the archetype P-type ATPase, SERCA (sarco(endo)plasmic reticulum Ca²⁺-ATPase), pumps contractile-dependent Ca²⁺ ions into the lumen of sarcoplasmic reticulum, which initiates myocyte relaxation and refills the sarcoplasmic reticulum in preparation for the next contraction. In cardiac muscle, SERCA is regulated by phospholamban (PLB), a small inhibitory phosphoprotein that decreases the Ca²⁺ affinity of SERCA and attenuates contractile strength. cAMP-dependent phosphorylation of PLB reverses Ca²⁺-ATPase inhibition with powerful contractile effects. Here we present the long sought crystal structure of the PLB-SERCA complex at 2.8-Å resolution. The structure was solved in the absence of Ca²⁺ in a novel detergent system employing alkyl mannosides. The structure shows PLB bound to a previously undescribed conformation of SERCA in which the Ca²⁺ binding sites are collapsed and devoid of divalent cations (E2-PLB). This new structure represents one of the key unsolved conformational states of SERCA and provides a structural explanation for how dephosphorylated PLB decreases Ca²⁺ affinity and depresses cardiac contractility.     

Human Menkes X-chromosome disease and the staphylococcal cadmium-resistance ATPase: a remarkable similarity in protein sequences

A search with the proposed amino acid translation product from the new ‘candidate gene’ for human Menkes disease against protein sequence libraries showed a remarkable similarity to that for the cadmium efflux ATPase from Staphylococcus aureus resistance plasmids. The Menkes sequence appears closer to the CadA Cd²⁺ sequence than to P-type ATPases from animal sources. Menkes syndrome is an X-chromosome invariably fatal disease that results from abberant copper metabolism. The gene that is defective in Menkes patients, i.e. the Menkes candidate gene, encodes a P-type ATPase, whose properties satisfactorily explain the phenotype of the disease. P-type ATPases are all cation pumps, either for uptake (e.g. the bacterial Kdp K⁺ ATPase), for efflux (e.g. the muscle sarcoplasmic reticulum Ca²⁺ ATPase), or for cation exchange (e.g. the animal cell Na⁺/K⁺ ATPase). These enzymes have a conserved aspartate residue that is transiently phosphorylated from ATP during the transport cycle, hence the name ‘P-type’ ATPase. The Menkes sequence shares with the staphylococcal CadA ATPase those regions common to all P-type ATPases and also an N-terminal dithiol region that was proposed to be a ‘metal-binding motif’. There are one or two copies of this motif in the available CadA sequences and six copies in the Menkes sequence.     

Interhelical hydrogen bonds in transmembrane region are important for function and stability of Ca2+-transporting ATPase

Ca²⁺-transporting adenosine triphosphatase (ATPase) of sarcoplasmic reticulum couples ATP hydrolysis with ion transport. Phosphorylation of the cytosolic region of the calcium-bound conformation (E1) of the protein leads to drastic conformational rearrangements of the transmembrane helices and the release of Ca²⁺. The resulting calcium-free conformation (E2) is less stable than the E1 form. The changes in van der Waals interactions and interhelical hydrogen bonding in the E1 and E2 conformations were compared. Conformational changes in the transmembrane region concomitant with the release of Ca²⁺ mainly affect the number of interhelical hydrogen bonds, which is reduced to half of that in E1 form, whereas the number of interhelical atomic pairwise contacts reflecting van der Waals interactions experience little change. The interhelical hydrogen bonds in Ca²⁺-transporting ATPase can be divided into two groups according to their roles: those that play a structural stabilizing role and those that are important for the correct geometry of the Ca²⁺ binding site. Interhelical hydrogen bonds in the transmembrane regions play important roles for the stability and specificity of helix-helix interactions in proteins where change of conformation is required for transport of ions or small molecules.     

Calcitonin gene-related peptide receptor independently stimulates 3′,5′-cyclic adenosine monophosphate and Ca2+ signaling pathways

The inhibition of ion transporting ATPases (Na⁺,K⁺-ATPase, Ca²⁺,Mg²⁺- and Ca²⁺-ATPase) by two amphiphilic drugs e.g. chlorpromazine (antipsychotic) and chloroquine (antimalarial) are found to be competitive in nature in vitro with respect to the substrate. Two binding sites - high and low affinity are found to exist on all the three ATPases toward these drugs as evident from the plot of F/F₀ vs. different drug concentrations of tryptophan fluorescence of the enzymes. Circular dichroism analysis suggest that binding of drugs to the high affinity site does not involve any change in conformation of ATPase molecules which occur only when drug binds to the low affinity sites. The drug binding sites and possible effect on conformational change of ATPase molecules of these two drugs have been described in this report.     

Constitutive activation of gastric H+,K+-ATPase by a single mutation.

In the reaction cycle of P-type ATPases, an acid-stable phosphorylated intermediate is formed which is present in an intracellularly located domain of the membrane-bound enzymes. In some of these ATPases, such as Na+,K+-ATPase and gastric H+, K+-ATPase, extracellular K+ ions stimulate the rate of dephosphorylation of this phosphorylated intermediate and so stimulate the ATPase activity. The mechanism by which extracellular K+ ions stimulate the dephosphorylation process is unresolved. Here we show that three mutants of gastric H+,K+-ATPase lacking a negative charge on residue 820, located in transmembrane segment six of the alpha-subunit, have a high SCH 28080-sensitive, but K+-insensitive ATPase activity. This high activity is caused by an increased 'spontaneous' rate of dephosphorylation of the phosphorylated intermediate. A mutant with an aspartic acid instead of a glutamic acid residue in position 820 showed hardly any ATPase activity in the absence of K+, but K+ ions stimulated ATPase activity and the dephosphorylation process. These findings indicate that the negative charge normally present on residue 820 inhibits the dephosphorylation process. K+ ions do not stimulate dephosphorylation of the phosphorylated intermediate directly, but act by neutralizing the inhibitory effect of a negative charge in the membrane.     

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.     

Structural features of cation transport ATPases

Several cation transport ATPases, sharing the common feature of a phosphorylated intermediate in the process of ATP utilization, are compared with respect to their subunit composition and amino acid sequence. The main component of these enzymes is a polypeptide chain of MW slightly exceeding 100,000, comprising an extramembranous globular head which is connected through a stalk to a membrane-bound region. With reference to the Ca²⁺ ATPase of sarcoplasmic reticulum, it is proposed that the catalytic (ATP binding and phosphorylation) domain resides in the extramembranous globular head, while cation binding occurs in the membrane region. Therefore, these two functional domains are separated by a distance of approximately 50 Å. Alignment of amino acid sequences reveals extensive homology in the isoforms of the same ATPases, but relatively little homology in different cation ATPases. On the other hand, all cation ATPases considered in this analysis retain a consensus sequence of high homology, spanning the distance between the phosphorylation site and the preceding transmembrane helix. It is proposed that this sequence provides long-range functional linkage between catalytic and cation-binding domains. Thereby, translocation of bound cation occurs through a channel formed by transmembrane helices linked to the phosphorylation site. Additional sequences at the carboxyl terminal provide regulatory domains in certain ATPases.     

Calcium and copper transport ATPases: analogies and diversities in transduction and signaling mechanisms

The calcium transport ATPase and the copper transport ATPase are members of the P-ATPase family and retain an analogous catalytic mechanism for ATP utilization, including intermediate phosphoryl transfer to a conserved aspartyl residue, vectorial displacement of bound cation, and final hydrolytic cleavage of Pi. Both ATPases undergo protein conformational changes concomitant with catalytic events. Yet, the two ATPases are prototypes of different features with regard to transduction and signaling mechanisms. The calcium ATPase resides stably on membranes delimiting cellular compartments, acquires free Ca²⁺ with high affinity on one side of the membrane, and releases the bound Ca²⁺ on the other side of the membrane to yield a high free Ca²⁺ gradient. These features are a basic requirement for cellular Ca²⁺ signaling mechanisms. On the other hand, the copper ATPase acquires copper through exchange with donor proteins, and undergoes intracellular trafficking to deliver copper to acceptor proteins. In addition to the cation transport site and the conserved aspartate undergoing catalytic phosphorylation, the copper ATPase has copper binding regulatory sites on a unique N-terminal protein extension, and has also serine residues undergoing kinase assisted phosphorylation. These additional features are involved in the mechanism of copper ATPase intracellular trafficking which is required to deliver copper to plasma membranes for extrusion, and to the trans-Golgi network for incorporation into metalloproteins. Isoform specific glyocosylation contributes to stabilization of ATP7A copper ATPase in plasma membranes.     

Structure and function of the P-type ATPases.

The P-type ATPases are integral membrane proteins that generate essential transmembrane ion gradients in virtually all living cells. The structures of two of these have recently been elucidated at a resolution of 8 A. When considered together with the large body of biochemical information that has accrued for these transporters and for enzymes in general, this new structural information is providing tantalizing insights regarding the molecular mechanism of active ion transport catalyzed by these proteins.     

E2P-like states of plasma membrane Ca2+‑ATPase characterization of vanadate and fluoride-stabilized phosphoenzyme analogues

The plasma membrane Ca²⁺?ATPase (PMCA) belongs to the family of P-type ATPases, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. The crystal structure of PMCA is currently lacking. Its abundance is approximately 0.1% of the total protein in the membrane, hampering efforts to produce suitable crystals for X-ray structure analysis. In this work we characterized the effect of beryllium fluoride (BeF_x), aluminium fluoride (AlF_x) and magnesium fluoride (MgF_x) on PMCA. These compounds are known inhibitors of P-type ATPases that stabilize E2P ground, E2·P phosphoryl transition and E2·P_i product states. Our results show that the phosphate analogues BeF_x, AlF_x and MgF_x inhibit PMCA Ca²⁺?ATPase activity, phosphatase activity and phosphorylation with high apparent affinity. Ca²⁺?ATPase inhibition by AlF_x and BeF_x depended on Mg²⁺ concentration indicating that this ion stabilizes the complex between these inhibitors and the enzyme. Low pH increases AlF_x and BeF_x but not MgF_x apparent affinity. Eosin fluorescent probe binds with high affinity to the nucleotide binding site of PMCA. The fluorescence of eosin decreases when fluoride complexes bind to PMCA indicating that the environment of the nucleotide binding site is less hydrophobic in E2P-like states. Finally, measuring the time course of E?→?E2P-like conformational change, we proposed a kinetic model for the binding of fluoride complexes and vanadate to PMCA.In summary, our results show that these fluoride complexes reveal different states of phosphorylated intermediates belonging to the mechanism of hydrolysis of ATP by the PMCA.     

P-type ATPases of eukaryotes and bacteria: Sequence analyses and construction of phylogenetic trees

The amino acid sequences of 47 P-type ATPases from several eukaryotic and bacterial kingdoms were divided into three structural segments based on individual hydropathy profiles. Each homologous segment was (1) multiply aligned and functionally evaluated, (2) statistically analyzed to determine the degrees of sequence similarity, and (3) used for the construction of parsimonious phylogenetic trees. The results show that all of the P-type ATPases analyzed comprise a single family with four major clusters correlating with their cation specificities and biological sources as follows: cluster 1: Ca²⁺-transporting ATPases; cluster 2: Na⁺- and gastric H⁺-ATPases; cluster 3: plasma membrane H⁺-translocating ATPases of plants, fungi, and lower eukaryotes; and cluster 4: all but one of the bacterial P-type ATPases (specific for K⁺, Cd²⁺, Cu²⁺ and an unknown cation). The one bacterial exception to this general pattern was the Mg²⁺-ATPase of Salmonella typhimurium, which clustered with the eukaryotic sequences. Although exceptions were noted, the similarities of the phylogenetic trees derived from the three segments analyzed led to the probability that the N-terminal segments 1 and the centrally localized segments 2 evolved from a single primordial ATPase which existed prior to the divergence of eukaryotes from prokaryotes. By contrast, the C-terminal segments 3 appear to be eukaryotic specific, are not found in similar form in any of the prokaryotic enzymes, and are not all demonstrably homologous among the eukaryotic enzymes. These C-terminal domains may therefore have either arisen after the divergence of eukaryotes from prokaryotes or exhibited more rapid sequence divergence than either segment 1 or 2, thus masking their common origin. The relative rates of evolutionary divergence for the three segments were determined to be segment 2 < segment 1 < segment 3. Correlative functional analyses of the most conserved regions of these ATPases, based on published site-specific mutagenesis data, provided preliminary evidence for their functional roles in the transport mechanism. Our studies define the structural and evolutionary relationships among the P-type ATPases. They should provide a guide for the design of future studies of structure-function relationships employing molecular genetic, biochemical, and biophysical techniques. Correspondence to: M.H. Saier, Jr.     

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.     

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.     

P-type ATPases as drug targets: Tools for medicine and science

P-type ATPases catalyze the selective active transport of ions like H⁺, Na⁺, K⁺, Ca²⁺, Zn²⁺, and Cu²⁺ across diverse biological membrane systems. Many members of the P-type ATPase protein family, such as the Na⁺,K⁺-, H⁺,K⁺-, Ca²⁺-, and H⁺-ATPases, are involved in the development of pathophysiological conditions or provide critical function to pathogens. Therefore, they seem to be promising targets for future drugs and novel antifungal agents and herbicides. Here, we review the current knowledge about P-type ATPase inhibitors and their present use as tools in science, medicine, and biotechnology. Recent structural information on a variety of P-type ATPase family members signifies that all P-type ATPases can be expected to share a similar basic structure and a similar basic machinery of ion transport. The ion transport pathway crossing the membrane lipid bilayer is constructed of two access channels leading from either side of the membrane to the ion binding sites at a central cavity. The selective opening and closure of the access channels allows vectorial access/release of ions from the binding sites. Recent structural information along with new homology modeling of diverse P-type ATPases in complex with known ligands demonstrate that the most proficient way for the development of efficient and selective drugs is to target their ion transport pathway.     

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.     

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.