Membrane topology of the p1258 CadA Cd(II)/Pb(II)/Zn(II)-translocating P-type ATPase

Plasmid pl258 carries the cadA gene that confers resistance to cadmium, lead, and zinc. CadA catalyzes ATP-dependent cadmium efflux from cells of Staphylococcus aureus. It is a member of the superfamily of P-type ATPases and belongs to the subfamily of soft metal ion pumps. In this study the membrane topology of this P-type ATPase was determined by constructing fusions with the topological reporter genes phoA or lacZ. A series of 44 C-terminal truncated CadAs were fused with one or the other reporter gene, and the activity of each chimeric protein was determined. In addition, the location of the first transmembrane segment was determined by immunoblot analysis. The results are consistent with the pl258 CadA ATPase having eight transmembrane segments. The first 109 residues is a cytosolic domain that includes the Cys(X)₂Cys motif that distinguishes soft metal ion-translocating P-type ATPases from their hard metal ion-translocating homologues. Another feature of soft metal ion P-type ATPases is the CysProCys motif, which is found in the sixth transmembrane segment of CadA. The phosphorylation site and ATP binding domain conserved in all P-type ATPases are situated within the large cytoplasmic loop between the sixth and seventh transmembrane segments.     

Characterization of a putative type IV aminophospholipid transporter P-type ATPase

The P-type ATPases comprise a well-studied family of proteins involved in the active transport of charged substrates across biological membranes. Starting from a mouse bone marrow-derived macrophage cDNA library and using a signal peptide trapping strategy, we identified a new P-type ATPase family member. We characterized the genomic structure of this gene, named Atp10d, as well as its human counterpart. The presence of P-type ATPase consensus motifs and phylogenetic analysis showed that this gene is a member of the type IV, putative amphipath transporters subfamily. We showed that this gene is expressed in kidney and placenta. We also found that the C57BL/6 strain carries a constitutive stop codon in the sequence of Atp10d exon 12, whereas 14 other inbred mouse strains show an uninterrupted reading frame at this location. This mutation in C57BL/6 should lead to a non-functional protein, suggesting that this gene may not be essential. We discuss the involvement of the Atp10d gene in the fat-prone phenotype of the C57BL/6 strain and its physical mapping within a QTL associated with HDL-cholesterol levels.     

Cloning, mapping and expression analysis of the sheep Wilson disease gene homologue

Copper homeostasis in mammals is maintained by the balance of dietary intake and copper excretion via the bile. Sheep have a variant copper phenotype and do not efficiently excrete copper by this mechanism, often resulting in excessive copper accumulation in the liver. The Wilson disease protein (ATP7B) is a copper transporting P-type ATPase that is responsible for the efflux of hepatic copper into the bile. To investigate the role of ATP7B in the sheep copper accumulation phenotype, the cDNA encoding the ovine homologue of ATP7B was isolated and sequenced and the gene was localised by fluorescence in situ hybridisation to chromosome 10. The 6.3 kb cDNA encoded a predicted protein of 1444 amino acids which included all of the functional domains characteristic of copper transporting P-type ATPases. ATP7B mRNA was expressed primarily in the liver with lower levels present in the intestine, hypothalamus and ovary. A splice variant of ATP7B mRNA, which was expressed in the liver and comprised approximately 10% of the total ATP7B mRNA pool, also was isolated. The results suggest that ATP7B is produced in the sheep and that the tendency to accumulate copper in the liver is not due to a gross alteration in the structure or expression of ATP7B.     

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.     

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.     

An extracellular loop of the human non-gastric H,K-ATPase alpha-subunit is involved in apical plasma membrane polarization.

The human non-gastric H,K-ATPase, ATP1AL1, belongs to the gene family of P-type ATPases. Consistent with their physiological roles in ion transport, members of this group, including the Na,KATPase and the gastric and non-gastric H,K-ATPases, are differentially polarized to either the basolateral or apical plasma membrane in epithelial cells. However, their polarized distribution is highly complex and depends on specific sorting signals or motifs which are recognized by the subcellular targeting machinery. For the gastric H,K-ATPase it has been suggested that the 4(th) transmembrane spanning domain (TM4) and its flanking regions induce conformational sorting motifs which direct the ion pump exclusively to the epithelial apical membrane. Here, we show in transfected Madin-Darby canine kidney (MDCK) cells that the related non-gastric H,KATPase, ATP1AL1, does contain similar sorting motifs in close proximity to TM4. A short extracellular loop between TM3 and TM4 is critical for this pump's apical delivery. A single point mutation in the corresponding region redirects ATP1AL1 to the basolateral membrane. In conclusion, our work provides further evidence that the cellular distribution of P-type ATPases is determined by conformational sorting motifs.     

Structural and functional insights of Wilson disease copper-transporting ATPase

Wilson disease is an autosomal recessive disorder of copper metabolism. The gene for this disorder has been cloned and identified to encode a copper-transporting ATPase (ATP7B), a member of a large family of cation transporters, the P-type ATPases. In addition to the core elements common to all P-type ATPases, the Wilson copper-transporting ATPase has a large cytoplasmic N-terminus comprised six heavy metal associated (HMA) domains, each of which contains the copper-binding sequence motif GMT/HCXXC. Extensive studies addressing the functional, regulatory, and structural aspects of heavy metal transport by heavy metal transporters in general, have offered great insights into copper transport by Wilson copper-transporting ATPase. The findings from these studies have been used together with homology modeling of the Wilson disease copper-transporting ATPases based on the X-ray structure of the sarcoplasmic reticulum (SR) calcium-ATPase, to present a hypothetical model of the mechanism of copper transport by copper-transporting ATPases.     

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.     

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.     

Structure of the ATP binding domain from the Archaeoglobus fulgidus Cu+-ATPase

The P-type ATPases translocate cations across membranes using the energy provided by ATP hydrolysis. CopA from Archaeoglobus fulgidus is a hyperthermophilic ATPase responsible for the cellular export of Cu+ and is a member of the heavy metal P1B-type ATPase subfamily, which includes the related Wilson and Menkes diseases proteins. The Cu+-ATPases are distinct from their P-type counter-parts in ion binding sequences, membrane topology, and the presence of cytoplasmic metal binding domains, suggesting that they employ alternate forms of regulation and novel mechanisms of ion transport. To gain insight into Cu+-ATPase function, the structure of the CopA ATP binding domain (ATPBD) was determined to 2.3 A resolution. Similar to other P-type ATPases, the ATPBD includes nucleotide binding (N-domain) and phosphorylation (P-domain) domains. The ATPBD adopts a closed conformation similar to the nucleotide-bound forms of the Ca2+-ATPase. The CopA ATPBD is much smaller and more compact, however, revealing the minimal elements required for ATP binding, hydrolysis, and enzyme phosphorylation. Structural comparisons to the AMP-PMP-bound form of the Escherichia coli K+-transporting Kdp-ATPase and to the Wilson disease protein N-domain indicate that the five conserved N-domain residues found in P1B-type ATPases, but not in the other families, most likely participate in ATP binding. By contrast, the P-domain includes several residues conserved among all P-type ATPases. Finally, the CopA ATPBD structure provides a basis for understanding the likely structural and functional effects of various mutations that lead to Wilson and Menkes diseases.     

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.     

Functional characterization of missense mutations in ATP7B: Wilson disease mutation or normal variant?

Wilson disease is an autosomal recessive disorder of copper transport that causes hepatic and/or neurological disease resulting from copper accumulation in the liver and brain. The protein defective in this disorder is a putative copper-transporting P-type ATPase, ATP7B. More than 100 mutations have been identified in the ATP7B gene of patients with Wilson disease. To determine the effect of Wilson disease missense mutations on ATP7B function, we have developed a yeast complementation assay based on the ability of ATP7B to complement the high-affinity iron-uptake deficiency of the yeast mutant ccc2. We characterized missense mutations found in the predicted membrane-spanning segments of ATP7B. Ten mutations have been made in the ATP7B cDNA by site-directed mutagenesis: five Wilson disease missense mutations, two mutations originally classified as possible disease-causing mutations, two putative ATP7B normal variants, and mutation of the cysteine-proline-cysteine (CPC) motif conserved in heavy-metal-transporting P-type ATPases. All seven putative Wilson disease mutants tested were able to at least partially complement ccc2 mutant yeast, indicating that they retain some ability to transport copper. One mutation was a temperature-sensitive mutation that was able to complement ccc2 mutant yeast at 30 degreesC but was unable to complement at 37 degreesC. Mutation of the CPC motif resulted in a nonfunctional protein, which demonstrates that this motif is essential for copper transport by ATP7B. Of the two putative ATP7B normal variants tested, one resulted in a nonfunctional protein, which suggests that it is a disease-causing mutation.     

Bacterial transition metal P(1B)-ATPases: transport mechanism and roles in virulence

P(1B)-type ATPases are polytopic membrane proteins that couple the hydrolysis of ATP to the efflux of cytoplasmic transition metals. This paper reviews recent progress in our understanding of the structure and function of these proteins in bacteria. These are members of the P-type superfamily of transport ATPases. Cu(+)-ATPases are the most frequently observed and best-characterized members of this group of transporters. However, bacterial genomes show diverse arrays of P(1B)-type ATPases with a range of substrates (Cu(+), Zn(2+), Co(2+)). Furthermore, because of the structural similarities among transitions metals, these proteins can also transport nonphysiological substrates (Cd(2+), Pb(2+), Au(+), Ag(+)). P(1B)-type ATPases have six or eight transmembrane segments (TM) with metal coordinating amino acids in three core TMs flanking the cytoplasmic domain responsible for ATP binding and hydrolysis. In addition, regulatory cytoplasmic metal binding domains are present in most P(1B)-type ATPases. Central to the transport mechanism is the binding of the uncomplexed metal to these proteins when cytoplasmic substrates are bound to chaperone and chelating molecules. Metal binding to regulatory sites is through a reversible metal exchange among chaperones and cytoplasmic metal binding domains. In contrast, the chaperone-mediated metal delivery to transport sites appears as a largely irreversible event. P(1B)-ATPases have two overarching physiological functions: to maintain cytoplasmic metal levels and to provide metals for the periplasmic assembly of metalloproteins. Recent studies have shown that both roles are critical for bacterial virulence, since P(1B)-ATPases appear key to overcome high phagosomal metal levels and are required for the assembly of periplasmic and secreted metalloproteins that are essential for survival in extreme oxidant environments.     

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.     

Molecular cloning of P-type ATPases on intracellular membranes of the marine alga Heterosigma akashiwo

Two cDNA clones (HAA13 and HAA1) which include conserved regions of genes of P-type ATPases were isolated from the marine alga Heterosigma akashiwo by a method that included the polymerase chain reaction. The longer cDNA (3286 bp), HAA13, consisted of an open reading frame that encoded a 106 kDa polypeptide of 977 amino acids with several possible transmembrane domains and conserved regions of eukaryotic P-type ATPases. One transmembrane domain had a leucine zipper structure. HAA1 was not a full-length gene (2054 bp) and lacked the 5 region, but it also included the conserved regions and putative transmembrane domains. Antibodies against the polypeptides encoded by HAA13 and HAA1 that have been expressed in Escherichia coli reacted with 100 kDa and 95 kDa polypeptides, respectively, on intracellular membranes of H. akashiwo cells. Immunostaining of H. akashiwo cells revealed that the HAA13 antigen was distributed on membranes around chloroplasts and the HAA1 antigen was located on small vesicles.     

Phytophthora nicotianae PnPMA1 encodes an atypical plasma membrane H+ -ATPase that is functional in yeast and developmentally regulated

PnPMA1, a gene encoding a putative P-type plasma membrane H(+)-ATPase, has been isolated by differential screening of a Phytophthora nicotianae germinated cyst cDNA library. PnPMA1 is differentially expressed during pathogen asexual development with a more than 10-fold increase in expression in germinated cysts, the stage at which plant infection is initiated, compared to vegetative or sporulating hyphae or motile zoospores. PnPMA1 proteins are encoded by two closely linked genes that have no introns and encode identical proteins having 1,068 amino acid residues and a molecular mass of 116.3kDa. PnPMA1 shows moderate identity (30-50%) to plant and fungal plasma membrane H(+)-ATPases and weak identity to other P-type cation-transporting ATPases. PnPMA1 contains all the catalytic domains characteristic of H(+)-ATPases but also has a distinct domain of approximately 155 amino acids that forms a putative cytoplasmic loop between transmembrane domains 8 and 9, a feature that is not present in PMA1 proteins from other organisms. Polyclonal antibodies raised against the 155 residue domain were shown by immunogold labelling to react with a protein in the plasma membrane of P. nicotianae germinated cysts but not with the plasma membrane of motile zoospores. Genetic complementation experiments demonstrated that the P. nicotianae PnPMA1 is functional in yeast, Saccharomyces cerevisiae.