Anticipating antiport in P-type ATPases

Cation-transporting P-type ATPases show a high degree of structural and functional homology. Nevertheless, for many members of this large family, the molecular mechanism of transport is unclear; namely, whether transport is electrogenic or not and if countertransport is involved remains to be established. In a few well-studied cases such as the Na(+)-K(+)-ATPase, plasma membrane Ca(2+) ATPase (PMCA) and sarcoplasmic reticulum Ca(2+) ATPase (SERCA) countertransport has been clearly demonstrated. New data based on the crystal structure of SERCA now strongly indicate that countertransport could be mandatory for all P-type ATPases. This concept should be verified for other known and for all newly characterized P-type ATPases.     

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.     

Copper transporting P-type ATPases and human disease

Copper transporting P-type ATPases, designated ATP7A and ATP7B, play an essential role in mammalian copper balance. Impaired intestinal transport of copper, resulting from mutations in the ATP7A gene, lead to Menkes disease in humans. Defects in a similar gene, the copper transporting ATPase ATP7B, result in Wilson disease. This ATP7B transporter has two functions: transport of copper into the plasma protein ceruloplasmin, and elimination of copper through the bile. Variants of ATP7B can be functionally assayed to identify defects in each of these functions. Tissue expression studies of the copper ATPases and their copper chaperone ATOX1 indicate that there is not complete overlap in expression. Other chaperones may be important for the transport of copper into ATP7A and ATP7B.     

Flexible P-type ATPases interacting with the membrane

Cation pumps and lipid flippases of the P-type ATPase family maintain electrochemical gradients and asymmetric lipid distributions across membranes, and offer significant insight of protein:membrane interactions. The sarcoplasmic reticulum Ca(2+)-ATPase features flexible and adaptive interactions with the surrounding membrane, while the Na(+),K(+)-ATPase complex is modulated by membrane components and a role for the γ-subunit as a stabilizer of a specific lipid interaction with the α-subunit has been proposed. The first crystal structure of a heavy-metal transporting ATPase shows a markedly amphipathic helix at the cytoplasmic membrane surface, highlighting this structure as a general motif of all P-type ATPases although with specialization to different membranes. Residues of central importance for the lipid flippase activity of the P4-type ATPase subfamily have been pinpointed by mutational studies, but the transport pathway and mechanism remain unknown.     

The multi-layered regulation of copper translocating P-type ATPases

The copper-translocating Menkes (ATP7A, MNK protein) and Wilson (ATP7B, WND protein) P-type ATPases are pivotal for copper (Cu) homeostasis, functioning in the biosynthetic incorporation of Cu into copper-dependent enzymes of the secretory pathway, Cu detoxification via Cu efflux, and specialized roles such as systemic Cu absorption (MNK) and Cu excretion (WND). Essential to these functions is their Cu and hormone-responsive distribution between the trans-Golgi network (TGN) and exocytic vesicles located at or proximal to the apical (WND) or basolateral (MNK) cell surface. Intriguingly, MNK and WND Cu-ATPases expressed in the same tissues perform distinct yet complementary roles. While intramolecular differences may specify their distinct roles, cellular signaling components are predicted to be critical for both differences and synergy between these enzymes. This review focuses on these mechanisms, including the cell signaling pathways that influence trafficking and bi-functionality of Cu-ATPases. Phosphorylation events are hypothesized to play a central role in Cu homeostasis, promoting multi-layered regulation and cross-talk between cuproenzymes and Cu-independent mechanisms.     

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.     

Charge transfer in P-type ATPases investigated on planar membranes

Planar lipid bilayers, e.g., black lipid membranes (BLM) and solid supported membranes (SSM), have been employed to investigate charge movements during the reaction cycle of P-type ATPases. The BLM/SSM method allows a direct measurement of the electrical currents generated by the cation transporter following chemical activation by a substrate concentration jump. The electrical current transients provides information about the reaction mechanism of the enzyme. In particular, the BLM/SSM technique allows identification of electrogenic steps which in turn may be used to localize ion translocation during the reaction cycle of the pump. In addition, using the high time resolution of the technique, especially when rapid activation via caged ATP is employed, rate constants of electrogenic and electroneutral steps can be determined. In the present review, we will discuss the main results obtained by the BLM and SSM methods and how they have contributed to unravel the transport mechanism of P-type ATPases.     

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.     

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.     

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.     

Mechanistic studies of phosphoserine phosphatase, an enzyme related to P-type ATPases

Phosphoserine phosphatase belongs to a new class of phosphotransferases forming an acylphosphate during catalysis and sharing three motifs with P-type ATPases and haloacid dehalogenases. The phosphorylated residue was identified as the first aspartate in the first motif (DXDXT) by mass spectrometry analysis of peptides derived from the phosphorylated enzyme treated with NaBH(4) or alkaline [(18)O]H(2)O. Incubation of native phosphoserine phosphatase with phosphoserine in [(18)O]H(2)O did not result in (18)O incorporation in residue Asp-20, indicating that the phosphoaspartate is hydrolyzed, as in P-type ATPases, by attack of the phosphorus atom. Mutagenesis studies bearing on conserved residues indicated that four conservative changes either did not affect (S109T) or caused a moderate decrease in activity (G178A, D179E, and D183E). Other mutations inactivated the enzyme by >80% (S109A and G180A) or even by >/=99% (D179N, D183N, K158A, and K158R). Mutations G178A and D179N decreased the affinity for phosphoserine, suggesting that these residues participate in the binding of the substrate. Mutations of Asp-179 decreased the affinity for Mg(2+), indicating that this residue interacts with the cation. Thus, investigated residues appear to play an important role in the reaction mechanism of phosphoserine phosphatase, as is known for equivalent residues in P-type ATPases and haloacid dehalogenases.     

Characterization of the P5 subfamily of P-type transport ATPases in mice

In mammals, the most poorly understood P-type ATPases are those of the P(5) subfamily. To begin characterization of the mammalian P(5)-ATPases, BLAST searches of DNA sequence databases were performed. Five genes were identified in the mouse, human, dog, and rat genomes, and the coding sequences of the mouse genes, termed Atp13a1-Atp13a5, were determined. The intron/exon organization of Atp13a1 differs entirely from those of Atp13a2-5, which are closely related. Amino acid sequence comparisons between the five mouse and two yeast P(5)-ATPases suggest that Atp13a1 is orthologous to the yeast Cod1 gene and that Atp13a2-5 are orthologous to yeast Yor291w. Northern blot analysis showed that Atp13a1, Atp13a2, and Atp13a3 mRNAs were expressed in all mouse tissues, whereas Atp13a4 and Atp13a5 mRNAs were restricted to brain and stomach. While the substrate specificity of these transporters is unknown, their importance is underscored by the presence of homologs in fish, insects, worms, and other eukaryotes.     

Two P-type ATPases are required for copper delivery in Arabidopsis thaliana chloroplasts

Copper delivery to the thylakoid lumen protein plastocyanin and the stromal enzyme Cu/Zn superoxide dismutase in chloroplasts is required for photosynthesis and oxidative stress protection. The copper delivery system in chloroplasts was characterized by analyzing the function of copper transporter genes in Arabidopsis thaliana. Two mutant alleles were identified of a previously uncharacterized gene, PAA2 (for P-type ATPase of Arabidopsis), which is required for efficient photosynthetic electron transport. PAA2 encodes a copper-transporting P-type ATPase with sequence similarity to PAA1, which functions in copper transport in chloroplasts. Both proteins localized to the chloroplast, as indicated by fusions to green fluorescent protein. The PAA1 fusions were found in the chloroplast periphery, whereas PAA2 fusions were localized in thylakoid membranes. The phenotypes of paa1 and paa2 mutants indicated that the two transporters have distinct functions: whereas both transporters are required for copper delivery to plastocyanin, copper delivery to the stroma is inhibited only in paa1 but not in paa2. The effects of paa1 and paa2 on superoxide dismutase isoform expression levels suggest that stromal copper levels regulate expression of the nuclear genes IRON SUPEROXIDE DISMUTASE1 and COPPER/ZINC SUPEROXIDE DISMUTASE2. A paa1 paa2 double mutant was seedling-lethal, underscoring the importance of copper to photosynthesis. We propose that PAA1 and PAA2 function sequentially in copper transport over the envelope and thylakoid membrane, respectively.     

Atx1-like chaperones and their cognate P-type ATPases: copper-binding and transfer

Copper is an essential yet toxic metal ion. To satisfy cellular requirements, while, at the same time, minimizing toxicity, complex systems of copper trafficking have evolved in all cell types. The best conserved and most widely distributed of these involve Atx1-like chaperones and P_1B-type ATPase transporters. Here, we discuss current understanding of how these chaperones bind Cu(I) and transfer it to the Atx1-like N-terminal domains of their cognate transporter.