Actuator Domain Research Articles

Relation -between Nanostructural Changes and Macroscopic Effects during Reversible Temperature-Memory Effect under Stress-Free Conditions in Semicrystalline Polymer Networks

ABSTRACTTemperature-memory effects in polymers under stress-free conditions are typically limited to one way effects. Recently, crosslinked polymer networks comprising crystallizable domains, which were capable of a reversible temperature-memory effect (rTME) under stress-free conditions, were introduced. The utilization of crystallizable actuator domains (AD) and shape determining domains (SD) where related to two different temperature ranges of a single broad melting temperature transition in case of rTME. In this study we investigated the nanostructure of crosslinked poly[ethylene-co-(vinyl acetate)] cPEVA capable of rTME in situ during actuation cycles utilizing X-ray scattering techniques and related the changes on the nanoscale to effects on the macroscopic scale. It was observed that 23% of SD obtained at a separation temperature of 75 °C gave the highest reversible strain and when exceeding 80 °C only isotropic crystallization occurred and no rTME was observed. Furthermore, distances between oriented crystalline lamellae correlated to the macroscopic actuation during heating-cooling cycles, exhibiting long-periods from 14 to 17 nm as function of temperature.

Second Transmembrane Helix (M2) and Long Range Coupling in Ca2+-ATPase

The actuator (A) domain of sarco(endo)plasmic reticulum Ca(2+)-ATPase not only plays a catalytic role but also undergoes large rotational movements that influence the distant transport sites through connections with transmembrane helices M1 and M2. Here we explore the importance of long helix M2 and its junction with the A domain by disrupting the helix structure and elongating with insertions of five glycine residues. Insertions into the membrane region of M2 and the top junctional segment impair Ca(2+) transport despite reasonable ATPase activity, indicating that they are uncoupled. These mutants fail to occlude Ca(2+). Those at the top segment also exhibited accelerated phosphoenzyme isomerization E1P → E2P. Insertions into the middle of M2 markedly accelerate E2P hydrolysis and cause strong resistance to inhibition by luminal Ca(2+). Insertions along almost the entire M2 region inhibit the dephosphorylated enzyme transition E2 → E1. The results pinpoint which parts of M2 control cytoplasm gating and which are critical for luminal gating at each stage in the transport cycle and suggest that proper gate function requires appropriate interactions, tension, and/or rigidity in the M2 region at appropriate times for coupling with A domain movements and catalysis.

Peptide-Based Approach to Study Cytosolic Domain Interactions in a Bacterial Copper-Transporting ATPase

Copper-transporting PIB-type ATPases (CopA-family) fulfill key roles in copper homeostasis by pumping copper across bacterial or intracellular and cytoplasmic eukaryotic membranes. Mutations of the copper-transporting ATPase gene cause Wilson and Menkes diseases. CopA consists of 8 transmembrane helices and three cytosolic domains; Nucleotide binding (N), Phosphorylation (P) and Actuator (A) domains. We have used synthetic peptides to identify minimal structural motifs and physical mechanisms of cytosolic domain interactions.Based on the crystal structure[1], we have designed decameric-peptides whose sequences are derived from the putative interaction site of the A-domain with the PN domain of CopA from Legionella pneumophila. The interaction of the synthetic A-domain peptide with the individually expressed PN domain was measured by Isothermal Titration Calorimetry(ITC), Circular Dichroism (CD), and stopped-flow fluorescence techniques. Preliminary ITC results show a favorable TΔS, indicating that binding of the peptide carrying the native sequence is driven by hydrophobic interactions with stoichiometry(N=1). Time-resolved fluorescence measurements with a tryptophan-carrying actuator peptide show association and dissociation rate constants with the PN domain of 22000M−2s−1 and 0.7M−1s−1, respectively. This corresponds to a Kd of the peptide for the PN-domain of 32μM. In the presence of the non-hydrolyzable nucleotide AMPPNP the best kinetic fit was obtained with rates of 20000M−2s−1 and 1.0M−1s−1, indicating an almost unaffected affinity of the peptide for the nucleotide-loaded PN-domain. In agreement with the kinetic and isothermal data, the deca-peptide also shifted the melting temperature of the PN domain from 330 to 332K as monitored by CD spectroscopy.In summary, we have good evidence that the actuator peptide binds in a predominantly hydrophobic mechanism to the PN domain in the 30-50μM Kd range with little dependence on the occupancy of the nucleotide binding site of the PN domain.1)Gourdon,P. et.al.2011 Nature475, 59.

Numerical Model for Electro‐Mechanical Analysis Based on Finite Element Method

AbstractIn this paper, we present a numerical procedure that can be used to model the electro‐mechanical coupled behavior of the dielectric actuator domain. The equation describing the electrostatical part is given by the reduced form of the Maxwell equation and the electrostatic potential [1]. The mechanical problem is described by the constitutive equations and equilibrium equations. Using the finite element method, this technique is to divide a whole problem into sub‐problems. The complexity of the original problem is therefore reduced by focusing only on the most relevant areas. A finite element analysis is then performed by applying the electrostatic Maxwell pressure as Neumann boundary conditions to compute the displacements. Once the displacement is computed, the electrostatic domain or the conductor is updated. Electrostatic analysis is performed on the updated geometry and the finite element method is then used to determine the change in potential due to geometric perturbations. Once the surface charge densities are known, the new electrostatic Maxwell pressure is computed. The mechanical and electrostatic analysis is repeated until an equilibrium state is computed. The procedure is demonstrated in the paper by the solution of some two‐dimensional and three‐dimensional problems. (© 2013 Wiley‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Involvement of Reactive Oxygen Species in a Feed-forward Mechanism of Na/K-ATPase-mediated Signaling Transduction

Cardiotonic steroids (such as ouabain) signaling through Na/K-ATPase regulate sodium reabsorption in the renal proximal tubule. We report here that reactive oxygen species are required to initiate ouabain-stimulated Na/K-ATPase·c-Src signaling. Pretreatment with the antioxidant N-acetyl-L-cysteine prevented ouabain-stimulated Na/K-ATPase·c-Src signaling, protein carbonylation, redistribution of Na/K-ATPase and sodium/proton exchanger isoform 3, and inhibition of active transepithelial (22)Na(+) transport. Disruption of the Na/K-ATPase·c-Src signaling complex attenuated ouabain-stimulated protein carbonylation. Ouabain-stimulated protein carbonylation is reversed after removal of ouabain, and this reversibility is largely independent of de novo protein synthesis and degradation by either the lysosome or the proteasome pathways. Furthermore, ouabain stimulated direct carbonylation of two amino acid residues in the actuator domain of the Na/K-ATPase α1 subunit. Taken together, the data indicate that carbonylation modification of the Na/K-ATPase α1 subunit is involved in a feed-forward mechanism of regulation of ouabain-mediated renal proximal tubule Na/K-ATPase signal transduction and subsequent sodium transport.

A plausible mechanism for the antimalarial activity of artemisinin: A computational approach.

Artemisinin constitutes the frontline treatment to aid rapid clearance of parasitaemia and quick resolution of malarial symptoms. However, the widespread promiscuity about its mechanism of action is baffling. There is no consensus about the biochemical target of artemisinin but recent studies implicate haem and PfATP6 (a calcium pump). We investigated the role of iron and artemisinin on PfATP6, in search of a plausible mechanism of action, via density functional theory calculations, docking and molecular dynamics simulations. Results suggest that artemisinin gets activated by iron which in turn inhibits PfATP6 by closing the phosphorylation, nucleotide binding and actuator domains leading to loss of function of PfATP6 of the parasite and its death. The mechanism elucidated here should help in the design of novel antimalarials.

ModulatoryATPbinding to the E2 state of maize plasma membrane H+‐ATPase indicated by the kinetics of vanadate inhibition

P-type ATPases, as major consumers of cellular ATP in eukaryotic cells, are characterized by the formation of a phosphorylated enzyme intermediate (E2P), a process that is allosterically coupled to translocation of cations against an electrochemical gradient. The catalytic cycle comprises binding of Mg-ATP at the nucleotide-binding domain, phosphorylation of the E1 state (E1), conformational transition to the E2P state, and dephosphorylation through the actuator domain and re-establishment of the E1 state. Recently, it has been suggested that, for several P-type ATPases, Mg-ATP binds to the phosphorylated enzyme, thereby accelerating the transition to the E1 state, before then becoming the enzyme's catalytic substrate. Here, we provide evidence supporting this viewpoint. We employed kinetic models based on steady-state kinetics in the presence and absence of the reversible inhibitor orthovanadate. Vanadate is generally considered to be a conformational probe that specifically binds to the E2 state, arresting the enzyme in a state analogous to the E2P state. Hydrolytic H(+) -ATPase activities were measured in inside-out plasma membrane vesicles isolated from roots and shoots of maize plants. For root enzymes, kinetic models of vanadate inhibition that allow simultaneous binding of Mg-ATP and vanadate to the same enzyme state were most plausible. For shoot enzymes, application of the competitive inhibitor Mg-free ATP attenuated vanadate inhibition, which is consistent with a model in which either Mg-free ATP or Mg-ATP is bound to the enzyme when vanadate binds. Therefore, data from roots and shoots indicate that binding of ATP species before transition to the E1 state plays an important role in the catalytic cycle of plant plasma membrane H(+) -ATPase.

Familial hemiplegic migraine mutations affect Na,K-ATPase domain interactions

Familial hemiplegic migraine (FHM) is a monogenic variant of migraine with aura. One of the three known causative genes, ATP1A2, which encodes the α2 isoform of Na,K-ATPase, causes FHM type 2 (FHM2). Over 50 FHM2 mutations have been reported, but most have not been characterized functionally. Here we study the molecular mechanism of Na,K-ATPase α2 missense mutations. Mutants E700K and P786L inactivate or strongly reduce enzyme activity. Glutamic acid 700 is located in the phosphorylation (P) domain and the mutation most likely disrupts the salt bridge with Lysine 35, thereby destabilizing the interaction with the actuator (A) domain. Mutants G900R and E902K are present in the extracellular loop at the interface of the α and β subunit. Both mutants likely hamper the interaction between these subunits and thereby decrease enzyme activity. Mutants E174K, R548C and R548H reduce the Na+ and increase the K+ affinity. Glutamic acid 174 is present in the A domain and might form a salt bridge with Lysine 432 in the nucleotide binding (N) domain, whereas Arginine 548, which is located in the N domain, forms a salt bridge with Glutamine 219 in the A domain. In the catalytic cycle, the interactions of the A and N domains affect the K+ and Na+ affinities, as observed with these mutants. Functional consequences were not observed for ATP1A2 mutations found in two sporadic hemiplegic migraine cases (Y9N and R879Q) and in migraine without aura (R51H and C702Y).

Roles of Long-range Electrostatic Domain Interactions and K+ in Phosphoenzyme Transition of Ca2+-ATPase

Sarcoplasmic reticulum Ca(2+)-ATPase couples the motions and rearrangements of three cytoplasmic domains (A, P, and N) with Ca(2+) transport. We explored the role of electrostatic force in the domain dynamics in a rate-limiting phosphoenzyme (EP) transition by a systematic approach combining electrostatic screening with salts, computer analysis of electric fields in crystal structures, and mutations. Low KCl concentration activated and increasing salt above 0.1 m inhibited the EP transition. A plot of the logarithm of the transition rate versus the square of the mean activity coefficient of the protein gave a linear relationship allowing division of the activation energy into an electrostatic component and a non-electrostatic component in which the screenable electrostatic forces are shielded by salt. Results show that the structural change in the transition is sterically restricted, but that strong electrostatic forces, when K(+) is specifically bound at the P domain, come into play to accelerate the reaction. Electric field analysis revealed long-range electrostatic interactions between the N and P domains around their hinge. Mutations of the residues directly involved and other charged residues at the hinge disrupted in parallel the electric field and the structural transition. Favorable electrostatics evidently provides a low energy path for the critical N domain motion toward the P domain, overcoming steric restriction. The systematic approach employed here is, in general, a powerful tool for understanding the structural mechanisms of enzymes.

Time Resolved FRET in the SR Ca-ATPase

We have detected structural dynamics of the sarcoplasmic reticulum Ca-ATPase (SERCA) using time-resolved fluorescence spectroscopy. The Ca-ATPase from fast-twitch skeletal muscle (SERCA1a isoform) was labeled with cyan fluorescent protein (CFP) at the N-terminus in the actuator domain (A) and fluorescein isothiocyanate (FITC) at Lys-515 in the nucleotide-binding domain (N). Time-resolved FRET was detected between CFP (donor in A domain) and FITC (acceptor in N domain) for SERCA in ligand-stabilized states, including calcium-free (E2), calcium-bound (E1), and actively-cycling phosphoenzyme (EP). Lifetime fitting and molecular modeling were used to interpret fluorescence decays, thereby identifying a dynamic distribution of structural states within the cytoplasmic headpiece of SERCA (i.e., A-N interdomain distance). FRET results were compared to distance predictions from x-ray crystallography. We propose a structural mechanism for ligand activation of SERCA. Acknowledgments: Spectroscopy was performed in the Biophysical Spectroscopy Center at the University of Minnesota, with assistance from Fluorescence Innovations, Inc. (Gregory Gillispie, President). This work was funded by NIH grants to DDT (R01 GM27906, P30 AR0507220, T32 AR007612).

Output Feedback Sliding Mode Control for a Stewart Platform With a Nonlinear Observer-Based Forward Kinematics Solution

In this paper, an observer-based forward kinematics solution of a 6-6 Stewart platform is proposed and this algorithm is applied to implement an output feedback sliding mode control. The conventional forward kinematics solutions take too much computational load or are too complex to be carried out in the online control scheme. The proposed nonlinear observer-based algorithm provides a simple method to obtain a real-time forward kinematics solution. With this solution, 6-degrees-of-freedom posture control of the moving platform can be achieved without installation of any external sensor after applying an output feedback control. In contrast with the conventional control scheme which aims to control individual leg length in actuator domain, the output feedback controller is proposed here to control the posture in Cartesian domain directly. The stability of the whole system is thoroughly proved to ensure convergence of the control errors. Simulations and experimental results are presented to validate the feasibility of the hereby proposed results.

De novo design of a synthetic riboswitch that regulates transcription termination

Riboswitches are regulatory RNA elements typically located in the 5′-untranslated region of certain mRNAs and control gene expression at the level of transcription or translation. These elements consist of a sensor and an adjacent actuator domain. The sensor usually is an aptamer that specifically interacts with a ligand. The actuator contains an intrinsic terminator or a ribosomal binding site for transcriptional or translational regulation, respectively. Ligand binding leads to structural rearrangements of the riboswitch and to presentation or masking of these regulatory elements. Based on this modular organization, riboswitches are an ideal target for constructing synthetic regulatory systems for gene expression. Although riboswitches for translational control have been designed successfully, attempts to construct synthetic elements regulating transcription have failed so far. Here, we present an in silico pipeline for the rational design of synthetic riboswitches that regulate gene expression at the transcriptional level. Using the well-characterized theophylline aptamer as sensor, we designed the actuator part as RNA sequences that can fold into functional intrinsic terminator structures. In the biochemical characterization, several of the designed constructs show ligand-dependent control of gene expression in Escherichia coli, demonstrating that it is possible to engineer riboswitches not only for translational but also for transcriptional regulation.

Pseudomyotonia in Romagnola cattle caused by novel ATP2A1mutations

BackgroundBovine congenital pseudomyotonia (PMT) is an impairment of muscle relaxation induced by exercise preventing animals from performing rapid movements. Forms of recessively inherited PMT have been described in different cattle breeds caused by two independent mutations in ATP2A1 encoding a skeletal-muscle Ca2+-ATPase (SERCA1). We observed symptoms of congenital PMT in four related Romagnola beef cattle from Italy and evaluated SERCA1 activity and scanned ATP2A1 for possible causative mutations.ResultsWe obtained four PMT affected Romagnola cattle and noted striking clinical similarities to the previously described PMT cases in other cattle breeds. The affected animals had a reduced SERCA1 activity in the sarcoplasmic reticulum. A single affected animal was homozygous for a novel complex variant in ATP2A1 exon 8 (c.[632 G>T; 857 G>T]). Three out of four cases were compound heterozygous for the newly identified exon 8 variant and the exon 6 variant c.491 G>A(p. Arg146Gly), which has previously been shown to cause PMT in Chianina cattle. Pedigree analysis showed that the exon 8 double mutation event dates back to at least 1978. Both nucleotide substitutions are predicted to alter the SERCA1 amino acid sequence (p.[(Gly211Val; Gly284Val)]), affect highly conserved residues, in particular the actuator domain of SERCA1.ConclusionClinical, biochemical and DNA analyses confirmed the initial hypothesis. We provide functional and genetic evidence that one novel and one previously described ATP2A1 mutation lead to a reduced SERCA1 activity in skeletal muscles and pseudomyotonia in affected Romagnola cattle. Selection against these mutations can now be used to eliminate the mutant alleles from the Romagnola breed.

P-type ATPases at a glance

P-type ATPases are a large family of integral membrane transporters that are of vital importance in all kingdoms of life. In eponymous distinction from the other main classes of transport ATPase – the FF1 (F-), the vacuolar (V-) and the ATP-binding cassette (ABC-) type – the P-type ATPases form

Sets of Local Entropy-Enthalpy Change Leads to Global Entropy-Enthalpy Change in SERCA

Major conformational changes are involved in the multi-step catalytic cycle of the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA) pump. The movement and rotation of the Actuator (A) domain is crucial for ion translocation. The A-domain is connected to the trans-membrane helices through linker regions. Mutational studies on the A-M3 linker region show that varying the length of this region causes significant changes in the rate of the conformational transitions (JBC (2009),284,12258-12265 ). The focus of this research is to study the impact of these mutations on the structural changes during the transition from E1 to E2, the behavior of the A-M3 linker region, and the overall rate of the conformational transitions. In order to achieve faster computation of transitions, we implemented the MARTINI coarse-grained protein and lipid model in CHARMM. We used Dynamic Importance Sampling (DIMS) to compute transitions from the E1 to the E2 state for both directions of each mutant in both coarse-grained and all atom models. Analysis of the transitions across mutants shows that the angle formed by A-P-N domains changes by up to 20 degrees with an increasing number of inserts into the A-M3 linker region. Estimates of barrier crossing time from the simulation and experimental values are highly correlated (R2=0.934). Quasiharmonic analysis on the domains, linkers and transmembrane helices show entropic changes between the mutants and compensation effects. Interaction energies of the same regions indicate entropic-enthalpic compensation. Further investigation of the end state simulations shows changes in the number of high density water sites around the A-M3 linker region across the mutants. The varying degree of the change in volume due to water sites across all the mutants indicates a ripple effect where the local entropy-enthaply changes translate to global entropy-enthalpy changes.

The transport mechanism of bacterial Cu+-ATPases: distinct efflux rates adapted to different function

Cu(+)-ATPases play a key role in bacterial Cu(+) homeostasis by participating in Cu(+) detoxification and cuproprotein assembly. Characterization of Archaeoglobus fulgidus CopA, a model protein within the subfamily of P(1B-1) type ATPases, has provided structural and mechanistic details on this group of transporters. Atomic resolution structures of cytoplasmic regulatory metal binding domains (MBDs) and catalytic actuator, phosphorylation, and nucleotide binding domains are available. These, in combination with whole protein structures resulting from cryo-electron microscopy analyses, have enabled the initial modeling of these transporters. Invariant residues in helixes 6, 7 and 8 form two transmembrane metal binding sites (TM-MBSs). These bind Cu(+) with high affinity in a trigonal planar geometry. The cytoplasmic Cu(+) chaperone CopZ transfers the metal directly to the TM-MBSs; however, loading both of the TM-MBSs requires binding of nucleotides to the enzyme. In agreement with the classical transport mechanism of P-type ATPases, occupancy of both transmembrane sites by cytoplasmic Cu(+) is a requirement for enzyme phosphorylation and subsequent transport into the periplasmic or extracellular milieus. Recent transport studies have shown that all Cu(+)-ATPases drive cytoplasmic Cu(+) efflux, albeit with quite different transport rates in tune with their various physiological roles. Archetypical Cu(+)-efflux pumps responsible for Cu(+) tolerance, like the Escherichia coli CopA, have turnover rates ten times higher than those involved in cuproprotein assembly (or alternative functions). This explains the incapability of the latter group to significantly contribute to the metal efflux required for survival in high copper environments.

Boundary-value problems in information theory of relay protection

The problems of constructing actuation domains of recognition modules of relay protection, both autonomous and as part of a group, are discussed and illustrated by examples. The group forms a multidimensional structure trained by imitation models of a protected object in controlled and alternative regimes.

Stable Structural Analog of Ca2+-ATPase ADP-insensitive Phosphoenzyme with Occluded Ca2+ Formed by Elongation of A-domain/M1′-linker and Beryllium Fluoride Binding

We have developed a stable analog for the ADP-insensitive phosphoenzyme intermediate with two occluded Ca(2+) at the transport sites (E2PCa(2)) of sarcoplasmic reticulum Ca(2+)-ATPase. This is normally a transient intermediate state during phosphoenzyme isomerization from the ADP-sensitive to ADP-insensitive form and Ca(2+) deocclusion/release to the lumen; E1PCa(2) --> E2PCa(2) --> E2P + 2Ca(2+). Stabilization was achieved by elongation of the Glu(40)-Ser(48) loop linking the Actuator domain and M1 (1st transmembrane helix) with four glycine insertions at Gly(46)/Lys(47) and by binding of beryllium fluoride (BeF(x)) to the phosphorylation site of the Ca(2+)-bound ATPase (E1Ca(2)). The complex E2Ca(2)xBeF(3)(-) was also produced by lumenal Ca(2+) binding to E2xBeF(3)(-) (E2P ground state analog) of the elongated linker mutant. The complex was stable for at least 1 week at 25 degrees C. Only BeF(x), but not AlF(x) or MgF(x), produced the E2PCa(2) structural analog. Complex formation required binding of Mg(2+), Mn(2+), or Ca(2+) at the catalytic Mg(2+) site. Results reveal that the phosphorylation product E1PCa(2) and the E2P ground state (but not the transition states) become competent to produce the E2PCa(2) transient state during forward and reverse phosphoenzyme isomerization. Thus, isomerization and lumenal Ca(2+) release processes are strictly coupled with the formation of the acylphosphate covalent bond at the catalytic site. Results also demonstrate the critical structural roles of the Glu(40)-Ser(48) linker and of Mg(2+) at the catalytic site in these processes.

Structure and interactions of the C‐terminal metal binding domain of Archaeoglobus fulgidus CopA

The Cu(+)-ATPase CopA from Archaeoglobus fulgidus belongs to the P(1B) family of the P-type ATPases. These integral membrane proteins couple the energy of ATP hydrolysis to heavy metal ion translocation across membranes. A defining feature of P(1B-1)-type ATPases is the presence of soluble metal binding domains at the N-terminus (N-MBDs). The N-MBDs exhibit a conserved ferredoxin-like fold, similar to that of soluble copper chaperones, and bind metal ions via a conserved CXXC motif. The N-MBDs enable Cu(+) regulation of turnover rates apparently through Cu-sensitive interactions with catalytic domains. A. fulgidus CopA is unusual in that it contains both an N-terminal MBD and a C-terminal MBD (C-MBD). The functional role of the unique C-MBD has not been established. Here, we report the crystal structure of the apo, oxidized C-MBD to 2.0 A resolution. In the structure, two C-MBD monomers form a domain-swapped dimer, which has not been observed previously for similar domains. In addition, the interaction of the C-MBD with the other cytoplasmic domains of CopA, the ATP binding domain (ATPBD) and actuator domain (A-domain), has been investigated. Interestingly, the C-MBD interacts specifically with both of these domains, independent of the presence of Cu(+) or nucleotides. These data reinforce the uniqueness of the C-MBD and suggest a distinct structural role for the C-MBD in CopA transport.

Cation transport coupled to ATP hydrolysis by the (Na, K)‐ATPase

An Adobe® animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na(+) and K(+) translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P(2c) -type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also known as an E(1) /E(2) -ATPase as it undergoes conformational changes between the E(1) and E(2) forms during the pumping cycle, altering the affinity and accessibility of the transmembrane ion-binding sites. The animation is based on Horisberger's scheme that incorporates the most recent significant findings to have improved our understanding of the (Na, K)-ATPase structure-function relationship. The movements of the various domains within the (Na, K)-ATPase α-subunit illustrate the conformational changes that occur during Na(+) and K(+) translocation across the membrane and emphasize involvement of the actuator, nucleotide, and phosphorylation domains, that is, the "core engine" of the pump, with respect to ATP binding, cation transport, and ADP and P(i) release.