Aspartyl Phosphate Research Articles

Phenotypic plasticity: The role of a phosphatase family Rap in the genetic regulation of Bacilli.

In the last two decades, an increasing number of bacterial species have been recognized that are able to generate a phenotypically diverse population that shares an identical genotype. This ability is dependent on a complex genetic regulatory network that includes cellular and environmental signals, as well as stochastic elements. Among Bacilli, a broadly distributed family of Rap (Response-regulator aspartyl phosphate) phosphatases is known to modulate the function of the main phenotypic heterogeneity regulators by controlling their phosphorylation. Even more, their related extracellular Phr (Phosphatase regulator) peptides function as signals, creating a cell-cell communication network that regulates the phenotypic development of the entire population. In this review, we examine the role that the Rap phosphatases and their Phr peptides play in the regulation of Bacillus subtilis phenotypic differentiation, and in other members of the Bacillus genus. We also highlight the contribution of these regulatory elements to the fitness of bacterial cells and mobile genetic elements, for example, prophages and conjugative vectors.

The Solvation of the E. coli CheY Phosphorylation Site Mapped by XFMS.

The Escherichia coli CheY protein belongs to a large bacterial response regulator superfamily. X-ray hydroxy radical foot-printing with mass spectroscopy (XFMS) has shown that allosteric activation of CheY by its motor target triggers a concerted internalization of aromatic sidechains. We reanalyzed the XFMS data to compare polar versus non-polar CheY residue positions. The polar residues around and including the 57D phosphorylated site had an elevated hydroxy radical reactivity. Bioinformatic measures revealed that a water-mediated hydrogen bond network connected this ring of residues with the central 57D. These residues solvated 57D to energetically stabilize the apo-CheY fold. The abundance of these reactive residues was reduced upon activation. This result was supported by the bioinformatics and consistent with the previously reported activation-induced increase in core hydrophobicity. It further illustrated XFMS detection of structural waters. Direct contacts between the ring residues and the phosphorylation site would stabilize the aspartyl phosphate. In addition, we report that the ring residue, 18R, is a constant central node in the 57D solvation network and that 18R non-polar substitutions determine CheY diversity as assessed by its evolutionary trace in bacteria with well-studied chemotaxis. These results showcase the importance of structured water dynamics for phosphorylation-mediated signal transduction.

Fission yeast Duf89 and Duf8901 are cobalt/nickel-dependent phosphatase–pyrophosphatases that act via a covalent aspartyl–phosphate intermediate

Domain of Unknown Function 89 (DUF89) proteins are metal-dependent phosphohydrolases. Exemplary DUF89 enzymes differ in their metal and phosphosubstrate preferences. Here, we interrogated the activities and structures of two DUF89 paralogs from fission yeast—Duf89 and Duf8901. We find that Duf89 and Duf8901 are cobalt/nickel-dependent phosphohydrolases adept at hydrolyzing p-nitrophenylphosphate and PPi. Crystal structures of metal-free Duf89 and Co2+-bound Duf8901 disclosed two enzyme conformations that differed with respect to the position of a three-helix module, which is either oriented away from the active site in Duf89 or forms a lid over the active site in Duf8901. Lid closure results in a 16 Å movement of Duf8901 Asp195, vis-à-vis Asp199 in Duf89, that brings Asp195 into contact with an octahedrally coordinated cobalt. Reaction of Duf8901 with BeCl2 and NaF in the presence of divalent cations Co2+, Ni2+, or Zn2+ generated covalent Duf8901-(Asp248)–beryllium trifluoride (BeF3)•Co2+, Duf8901-(Asp248)–BeF3•Ni2+, or Duf8901-(Asp248)–BeF3•Zn2+ adducts, the structures of which suggest a two-step catalytic mechanism via formation and hydrolysis of an enzyme-(aspartyl)–phosphate intermediate. Alanine mutations of Duf8901 Asp248, Asn249, Lys401, Asp286, and Asp195 that interact with BeF3•Co2+ squelched p-nitrophenylphosphatase activity. A 1.8 Å structure of a Duf8901-(Asp248)–AlF4–OH2•Co2+ transition-state mimetic suggests an associative mechanism in which Asp195 and Asp363 orient and activate the water nucleophile. Whereas deletion of the duf89 gene elicited a phenotype in which expression of phosphate homeostasis gene pho1 was derepressed, deleting duf8901 did not, thereby hinting that the DUF89 paralogs have distinct functional repertoires in vivo.

Serine phosphorylation regulates the P-type potassium pump KdpFABC.

KdpFABC is an ATP-dependent K+ pump that ensures bacterial survival in K+-deficient environments. Whereas transcriptional activation of kdpFABC expression is well studied, a mechanism for down-regulation when K+ levels are restored has not been described. Here, we show that KdpFABC is inhibited when cells return to a K+-rich environment. The mechanism of inhibition involves phosphorylation of Ser162 on KdpB, which can be reversed in vitro by treatment with serine phosphatase. Mutating Ser162 to Alanine produces constitutive activity, whereas the phosphomimetic Ser162Asp mutation inactivates the pump. Analyses of the transport cycle show that serine phosphorylation abolishes the K+-dependence of ATP hydrolysis and blocks the catalytic cycle after formation of the aspartyl phosphate intermediate (E1~P). This regulatory mechanism is unique amongst P-type pumps and this study furthers our understanding of how bacteria control potassium homeostasis to maintain cell volume and osmotic potential.

The KdpFABC complex - K+ transport against all odds.

In bacteria, K+ is used to maintain cell volume and osmotic potential. Homeostasis normally involves a network of constitutively expressed transport systems, but in K+ deficient environments, the KdpFABC complex uses ATP to pump K+ into the cell. This complex appears to be a hybrid of two types of transporters, with KdpA descending from the superfamily of K+ transporters and KdpB belonging to the superfamily of P-type ATPases. Studies of enzymatic activity documented a catalytic cycle with hallmarks of classical P-type ATPases and studies of ion transport indicated that K+ import into the cytosol occurred in the second half of this cycle in conjunction with hydrolysis of an aspartyl phosphate intermediate. Atomic structures of the KdpFABC complex from X-ray crystallography and cryo-EM have recently revealed conformations before and after formation of this aspartyl phosphate that appear to contradict the functional studies. Specifically, structural comparisons with the archetypal P-type ATPase, SERCA, suggest that K+ transport occurs in the first half of the cycle, accompanying formation of the aspartyl phosphate. Further controversy has arisen regarding the path by which K+ crosses the membrane. The X-ray structure supports the conventional view that KdpA provides the conduit, whereas cryo-EM structures suggest that K+ moves from KdpA through a long, intramembrane tunnel to reach canonical ion binding sites in KdpB from which they are released to the cytosol. This review discusses evidence supporting these contradictory models and identifies key experiments needed to resolve discrepancies and produce a unified model for this fascinating mechanistic hybrid.

Hydrolysis of the E2P Phosphoenzyme of the Ca2+-ATPase: A Theoretical Study

Dephosphorylation of the E2P phosphoenzyme intermediate of the sarcoplasmic reticulum Ca(2+)-ATPase was studied using density functional theory. The hydrolysis reaction proceeds via a nucleophilic attack on the phosphorylated residue Asp351 by a water molecule, which is positioned by the nearby residue Glu183 acting as a base. The activation barrier was calculated to be 14.3 kcal/mol, which agrees well with values of 15-17 kcal/mol derived from experimentally observed rates. The optimized structure of the transition state reveals considerable bond breakage between phosphorus and the Asp351 oxygen (distance 2.19 Å) and little bond formation to the attacking water oxygen (distance 2.26 Å). Upon formation of the singly protonated phosphate product, Glu183 becomes protonated. The bridging aspartyl phosphate oxygen approaches Lys684 progressively when proceeding from the reactant state (distance 1.94 Å) via the transition state (1.78 Å) to the product state (1.58 Å). This stabilizes the negative charge that develops on the leaving group. The reaction was calculated to be slightly endergonic (+0.9 kcal/mol) and therefore reversible, in line with experimental findings. It is catalyzed by a preorganized active site with little movement of the nonreacting groups except for a rotation of Thr625 toward the phosphate group.

A Plasmid-Encoded Phosphatase Regulates Bacillus subtilis Biofilm Architecture, Sporulation, and Genetic Competence

Bacillus subtilis biofilm formation is tightly regulated by elaborate signaling pathways. In contrast to domesticated lab strains of B. subtilis which form smooth, essentially featureless colonies, undomesticated strains such as NCIB 3610 form architecturally complex biofilms. NCIB 3610 also contains an 80-kb plasmid absent from laboratory strains, and mutations in a plasmid-encoded homolog of a Rap protein, RapP, caused a hyperrugose biofilm phenotype. Here we explored the role of rapP phrP in biofilm formation. We found that RapP is a phosphatase that dephosphorylates the intermediate response regulator Spo0F. RapP appears to employ a catalytic glutamate to dephosphorylate the Spo0F aspartyl phosphate, and the implications of the RapP catalytic glutamate are discussed. In addition to regulating B. subtilis biofilm formation, we found that RapP regulates sporulation and genetic competence as a result of its ability to dephosphorylate Spo0F. Interestingly, while rap phr gene cassettes routinely form regulatory pairs; i.e., the mature phr gene product inhibits the activity of the rap gene product, the phrP gene product did not inhibit RapP activity in our assays. RapP activity was, however, inhibited by PhrH in vivo but not in vitro. Additional genetic analysis suggests that RapP is directly inhibited by peptide binding. We speculate that PhrH could be subject to posttranslational modification in vivo and directly inhibit RapP activity or, more likely, PhrH upregulates the expression of a peptide that, in turn, directly binds to RapP and inhibits its Spo0F phosphatase activity.

Near attack conformers dominate β-phosphoglucomutase complexes where geometry and charge distribution reflect those of substrate

Experimental observations of fluoromagnesate and fluoroaluminate complexes of β-phosphoglucomutase (β-PGM) have demonstrated the importance of charge balance in transition-state stabilization for phosphoryl transfer enzymes. Here, direct observations of ground-state analog complexes of β-PGM involving trifluoroberyllate establish that when the geometry and charge distribution closely match those of the substrate, the distribution of conformers in solution and in the crystal predominantly places the reacting centers in van der Waals proximity. Importantly, two variants are found, both of which satisfy the criteria for near attack conformers. In one variant, the aspartate general base for the reaction is remote from the nucleophile. The nucleophile remains protonated and forms a nonproductive hydrogen bond to the phosphate surrogate. In the other variant, the general base forms a hydrogen bond to the nucleophile that is now correctly orientated for the chemical transfer step. By contrast, in the absence of substrate, the solvent surrounding the phosphate surrogate is arranged to disfavor nucleophilic attack by water. Taken together, the trifluoroberyllate complexes of β-PGM provide a picture of how the enzyme is able to organize itself for the chemical step in catalysis through the population of intermediates that respond to increasing proximity of the nucleophile. These experimental observations show how the enzyme is capable of stabilizing the reaction pathway toward the transition state and also of minimizing unproductive catalysis of aspartyl phosphate hydrolysis.

Influence of the Molecular Environment on Phosphorylated Amino Acid Models: A Density Functional Theory Study

A protein environment can affect the structure and charge distribution of substrate molecules. Here, the structure and partial charges were studied for different phosphorylated amino acid models in varying environments using density functional theory. The three systems investigated, acetyl phosphate, methyl phosphate, and p-tolyl phosphate are representative models for aspartyl phosphate, serine or threonine phosphate, and tyrosine phosphate, respectively. Combined with the CPCM continuum model, explicit HF and H(2)O molecules were added in order to model environmental effects and interactions that may occur in a protein matrix. We show how the different interactions affect the scissile P-O(R) bond and that the elongation can be explained by an anomeric effect. An increasing scissile bond length will result in transfer of negative charge to the leaving group and in a widening of the angle between the terminal oxygens of the phosphate molecule, features that can expose the phosphate group to attacking nucleophiles. Lastly, calculations were performed on the active site of the Ca(2+)-ATPase E2P intermediate, which provide an example of how a protein environment facilitates the formation of a destabilized ground state.

Biochemical Characterization of AtHMA6/PAA1, a Chloroplast Envelope Cu(I)-ATPase

Copper is an essential plant micronutrient playing key roles in cellular processes, among them photosynthesis. In Arabidopsis thaliana, copper delivery to chloroplasts, mainly studied by genetic approaches, is thought to involve two P(IB)-type ATPases: AtHMA1 and AtHMA6/PAA1. The lack of biochemical characterization of AtHMA1 and PAA1, and more generally of plant P(IB)-type ATPases, is due to the difficulty of getting high amounts of these membrane proteins in an active form, either from their native environment or after expression in heterologous systems. In this study, we report the first biochemical characterization of PAA1, a plant copper-transporting ATPase. PAA1 produced in Lactococcus lactis is active, forming an aspartyl phosphate intermediate in the presence of ATP and the adequate metal ion. PAA1 can also be phosphorylated using inorganic phosphate in the absence of transition metal. Both phosphorylation types allowed us to demonstrate that PAA1 is activated by monovalent copper ions (and to a lower extent by silver ions) with an apparent affinity in the micromolar range. In agreement with these biochemical data, we also demonstrate that when expressed in yeast, PAA1 induces increased sensitivities to copper and silver. These data provide the first enzymatic characterization of a P(IB-1)-type plant ATPase and clearly identify PAA1 as a high affinity Cu(I) transporter of the chloroplast envelope.

Involvement of Protein Kinase D in Expression and Trafficking of ATP7B (Copper ATPase)

ATP7B is a P-type ATPase involved in copper transport and homeostasis. In experiments with microsomes isolated from COS-1 cells or HepG2 hepatocytes sustaining ATP7B heterologous expression, we found that ATP7B utilization of ATP includes autophosphorylation of an aspartyl residue serving as ATPase catalytic intermediate as well as phosphorylation of serine residues by protein kinase D (PKD). The latter was abolished by specific PKD inhibition with CID755673. The presence of PKD protein in the microsomal fraction was demonstrated by Western blotting. PKD is a serine/threonine kinase that associates with the trans-Golgi network, regulating fission of transport carriers destined to the cell surface. Parallel studies on cultured cells showed that nascent WT ATP7B transits to the Golgi complex where it undergoes serine phosphorylation by PKD. Misfolded ATP7B protein (especially if subjected to deletions) underwent proteasome-mediated degradation, which provides effective quality control. Inhibition of proteasome-mediated degradation with MG132 yielded additional, but nonfunctional protein. On the other hand, serine phosphorylation protected WT ATP7B from degradation. Protection was enhanced by PKD activation with phorbol esters and limited by PKD inhibition with CID75673. As a final step, phosphorylated ATP7B was transferred from the Golgi complex to cytosolic trafficking vesicles. Phosphorylation and trafficking were completely prevented by mutations of critical copper binding sites, demonstrating copper dependence of both PKD-assisted phosphorylation and trafficking. ATP7B trafficking was markedly reduced by the Ser-478/481/1121/1453 to Ala mutation. We conclude that PKD plays a key role in copper-dependent serine phosphorylation, permitting high levels of ATP7B protein expression and trafficking.

Functional Role for a Conserved Aspartate in the Spo0E Signature Motif Involved in the Dephosphorylation of the Bacillus subtilis Sporulation Regulator Spo0A

Sporulation is a complex developmental system characterizing Gram-positive bacteria of the genus Bacillus and Clostridium. In Bacillus subtilis the phosphorelay signal transduction system regulates the initiation of sporulation by integrating a myriad of positive and negative signals through the action of histidine sensor kinases and aspartyl phosphate phosphatases. The Spo0E family of phosphatases dephosphorylates the Spo0A response regulator and transcription factor of the phosphorelay. In this study we analyzed the role of the Spo0E signature motif in protein activity. This family is characterized by a conserved signature motif centered around the sequence "SQELD." Alanine scanning mutagenesis was carried out on the T(35)IXXSQ ELDCLI(46) residues of B. subtilis Spo0E and in vivo and in vitro activities were analyzed. The ability of the mutant proteins to interact with Spo0A approximately P was assayed by fluorescence resonance energy transfer spectroscopy. The results suggested that aspartate 43 has a critical role in Spo0E catalytic activity, whereas the other residues have a role in protein conformation and/or interaction with Spo0A. Residues Thr(35) and Cys(44) did not seem to have any critical functional or structural role. We propose that Asp(43) of Spo0E may function in a manner similar to the one proposed for the catalytic mechanisms of nucleotidase members of the haloacid dehalogenase family. These proteins use an aspartyl nucleophile as their common catalytic strategy and the active site of haloacid dehalogenase proteins shares a common geometry and identity of conserved amino acids with the active site of response regulators ( Ridder, I. S., and Dijkstra, B. W. (1999) Biochem. J. 339, 223-226 ).

Complications of Coinfection with Babesia and Lyme Disease After Splenectomy

<h3>Abstract</h3> KdpFABC is an ATP-dependent K⁺ pump that ensures bacterial survival in K⁺-deficient environments. Whereas transcriptional regulation of kdpFABC expression is well studied, a mechanism for regulating the pump when K⁺ levels are restored has not been described. Here we show that KdpFABC is inhibited by serine phosphorylation when cells return to a K⁺-rich environment. The mechanism of inhibition involves phosphorylation of Ser162 on KdpB, which is reversed by serine phosphatase. Mutating Ser162 to Alanine produces constitutive activity, whereas the phosphomimetic Ser162Asp mutation inactivates the pump. Analyses of partial reactions in the transport cycle show that serine phosphorylation uncouples the pump and blocks the cycle after formation of the catalytic aspartyl phosphate intermediate (E1~P). Molecular dynamics simulations show that serine phosphorylation affects domain dynamics that explain the uncoupling. This regulatory mechanism, unique amongst P-type pumps, furthers our understanding of how bacteria control potassium homeostasis to maintain cell volume and osmotic potential.

Negative Regulation ofBacillus anthracisSporulation by the Spo0E Family of Phosphatases

The initiation of sporulation in Bacillus species is controlled by the phosphorelay signal transduction system. Multiple regulatory elements act on the phosphorelay to modulate the level of protein phosphorylation in response to cellular, environmental, and metabolic signals. In Bacillus anthracis nine possible histidine sensor kinases can positively activate the system, while two response regulator aspartyl phosphate phosphatases of the Rap family negatively impact the pathway by dephosphorylating the Spo0F intermediate response regulator. In this study, we have characterized the B. anthracis members of the Spo0E family of phosphatases that specifically dephosphorylate the Spo0A response regulator of the phosphorelay and master regulator of sporulation. The products of four genes were able to promote the dephosphorylation of Spo0A approximately P in vitro. The overexpression of two of these B. anthracis Spo0E-like proteins from a multicopy vector consistently resulted in a sporulation-deficient phenotype. A third gene was found to be not transcribed in vivo. A fourth gene encoded a prematurely truncated protein due to a base pair deletion that nevertheless was subject to translational frameshift repair in an Escherichia coli protein expression system. A fifth Spo0E-like protein has been structurally and functionally characterized as a phosphatase of Spo0A approximately P by R. N. Grenha et al. (J. Biol. Chem. 281:37993-38003, 2006). We propose that these proteins may contribute to maintain B. anthracis in the transition phase of growth during an active infection and therefore contribute to the virulence of this organism.

Potassium as an Intrinsic Uncoupler of the Plasma Membrane H+-ATPase

The plant plasma membrane proton pump (H(+)-ATPase) is stimulated by potassium, but it has remained unclear whether potassium is actually transported by the pump or whether it serves other roles. We now show that K(+) is bound to the proton pump at a site involving Asp(617) in the cytoplasmic phosphorylation domain, from where it is unlikely to be transported. Binding of K(+) to this site can induce dephosphorylation of the phosphorylated E(1)P reaction cycle intermediate by a mechanism involving Glu(184) in the conserved TGES motif of the pump actuator domain. Our data identify K(+) as an intrinsic uncoupler of the proton pump and suggest a mechanism for control of the H(+)/ATP coupling ratio. K(+)-induced dephosphorylation of E(1)P may serve regulatory purposes and play a role in negative regulation of the transmembrane electrochemical gradient under cellular conditions where E(1)P is accumulating.

Conformational Cycling in β-Phosphoglucomutase Catalysis: Reorientation of the β-d-Glucose 1,6-(Bis)phosphate Intermediate

Activated Lactococcus lactis beta-phosphoglucomutase (betaPGM) catalyzes the conversion of beta-d-glucose 1-phosphate (betaG1P) derived from maltose to beta-d-glucose 6-phosphate (G6P). Activation requires Mg(2+) binding and phosphorylation of the active site residue Asp8. Initial velocity techniques were used to define the steady-state kinetic constants k(cat) = 177 +/- 9 s(-)(1), K(m) = 49 +/- 4 microM for the substrate betaG1P and K(m) = 6.5 +/- 0.7 microM for the activator beta-d-glucose 1,6-bisphosphate (betaG1,6bisP). The observed transient accumulation of [(14)C]betaG1,6bisP (12% at approximately 0.1 s) in the single turnover reaction carried out with excess betaPGM (40 microM) and limiting [(14)C]betaG1P (5 microM) and betaG1,6bisP (5 microM) supported the role of betaG1,6bisP as a reaction intermediate in the conversion of the betaG1P to G6P. Single turnover reactions of [(14)C]betaG1,6bisP with excess betaPGM were carried out to demonstrate that phosphoryl transfer rather than ligand binding is rate-limiting and to show that the betaG1,6bisP binds to the active site in two different orientations (one positioning the C(1)phosphoryl group for reaction with Asp8, and the other orientation positioning the C(6)phosphoryl group for reaction with Asp8) with roughly the same efficiency. Single turnover reactions carried out with betaPGM, [(14)C]betaG1P, and unlabeled betaG1,6bisP demonstrated complete exchange of label to the betaG1,6bisP during the catalytic cycle. Thus, the reorientation of the betaG1,6bisP intermediate that is required to complete the catalytic cycle occurs by diffusion into solvent followed by binding in the opposite orientation. Published X-ray structures of betaG1P suggest that the reorientation and phosphoryl transfer from betaG1,6bisP occur by conformational cycling of the enzyme between the active site open and closed forms via cap domain movement. Last, the equilibrium ratio of betaG1,6bisP to betaG1P plus G6P was examined to evidence a significant stabilization of betaPGM aspartyl phosphate.

FTIR studies on the bond properties of the aspartyl phosphate moiety of the Ca2+–ATPase

As part of our work to determine the bond properties of the aspartyl phosphate moiety of the Ca(2+)-ATPase (SERCA1a) phosphoenzymes, we analyzed Morse potentials of the bridging P-O bond as well as C=O bond strengths for the model compound acetyl phosphate and the two phosphoenzyme intermediates Ca(2)E1P and E2P. Reaction-induced infrared difference spectroscopy was used and a carbonyl band of E2P at 1708 cm(-1) in the presence of mM Mg(2+) was tentatively assigned to the carbonyl group of phosphorylated Asp(351) because of its sensitivity to divalent cations. This band is found at 1716 cm(-1) with mM Ca(2+), for Ca(2)E1P at 1717 cm(-1) with Mg(2+), and at 1719 cm(-1) with Ca(2+) and at 1718 cm(-1) for acetyl phosphate in the absence of divalent cations. The similar band positions indicate similar strengths of interaction of the carbonyl oxygen in acetyl phosphate and the two phosphoenzymes. Together with information on the P-O bond strengths, this implies that the bridging oxygen exerts stronger interactions in the phosphoenzymes than in acetyl phosphate.

Interactions of Phosphate Groups of ATP and Aspartyl Phosphate with the Sarcoplasmic Reticulum Ca 2+-ATPase: An FTIR Study

Phosphate binding to the sarcoplasmic reticulum Ca 2+-ATPase was studied by time-resolved Fourier transform infrared spectroscopy with ATP and isotopically labeled ATP ([ β- 18O 2, βγ- 18O]ATP and [ γ- 18O 3]ATP). Isotopic substitution identified several bands that can be assigned to phosphate groups of bound ATP: bands at 1260, 1207, 1145, 1110, and 1085 cm −1 are affected by labeling of the β-phosphate, bands likely near 1154, and 1098–1089 cm −1 are affected by γ-phosphate labeling. The findings indicate that the strength of interactions of β- and γ- phosphate with the protein are similar to those in aqueous solution. Two bands, at 1175 and 1113 cm −1, were identified for the phosphate group of the ADP-sensitive phosphoenzyme Ca 2E1P. They indicate terminal and bridging P-O bond strengths that are intermediate between those of ADP-insensitive phosphoenzyme E2P and the model compound acetyl phosphate in water. The bridging bond of Ca 2E1P is weaker than for acetyl phosphate, which will facilitate phosphate transfer to ADP, but is stronger than for E2P, which will make the Ca 2E1P phosphate less susceptible to attack by water.

Reconstruction of the Complete Ouabain-binding Pocket of Na,K-ATPase in Gastric H,K-ATPase by Substitution of Only Seven Amino Acids

Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na,K-ATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we started from the fact that a closely related enzyme, the gastric H,K-ATPase, does not bind glycosides like ouabain. Previously, we showed that a chimera of these two enzymes, in which only the M3-M4 and M5-M6 hairpins were of Na,K-ATPase, bound ouabain with high affinity (Koenderink, J. B., Hermsen, H. P. H., Swarts, H. G. P., Willems, P. H. G. M., and De Pont, J. J. H. H. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11209-11214). We also demonstrated that only three amino acids (Phe(783), Thr(797), and Asp(804)) present in the M5-M6 hairpin of Na,K-ATPase were sufficient to confer high affinity ouabain binding to a chimera which contained in addition the M3-M4 hairpin of Na,K-ATPase (Qiu, L. Y., Koenderink, J. B., Swarts, H. G., Willems, P. H., and De Pont, J. J. H. H. M. (2003) J. Biol. Chem. 278, 47240-47244). To further pinpoint the ouabain-binding site here we used a chimera-based loss-of-function strategy and identified four amino acids (Glu(312), Val(314), Ile(315), Gly(319)), all present in M4, as being important for ouabain binding. In a final gain-of-function study we showed that a gastric H,K-ATPase that contained Glu(312), Val(314), Ile(315), Gly(319), Phe(783), Thr(797), and Asp(804) of Na,K-ATPase bound ouabain with the same affinity as the native enzyme. Based on the E(2)P crystal structure of Ca(2+)-ATPase we constructed a homology model for the ouabain-binding site of Na,K-ATPase involving all seven amino acids as well as several earlier postulated amino acids.

Structural Studies of a Stabilized Phosphoenzyme Intermediate of Ca2+-ATPase

Ca(2+)-ATPase belongs to the family of P-type ATPases and maintains low concentrations of intracellular Ca(2+). Its reaction cycle consists of four main intermediates that alternate ion binding in the transmembrane domain with phosphorylation of an aspartate residue in a cytoplasmic domain. Previous work characterized an ultrastable phosphoenzyme produced first by labeling with fluorescein isothiocyanate, then by allowing this labeled enzyme to establish a maximal Ca(2+) gradient, and finally by removing Ca(2+) from the solution. This phosphoenzyme is characterized by very low fluorescence and has specific enzymatic properties suggesting the existence of a high energy phosphoryl bond. To study the structural properties of this phosphoenzyme, we used cryoelectron microscopy of two-dimensional crystals formed in the presence of decavanadate and determined the structure at 8-A resolution. To our surprise we found that at this resolution the low fluorescence phosphoenzyme had a structure similar to that of the native enzyme crystallized under equivalent conditions. We went on to use glutaraldehyde cross-linking and proteolysis for independent structural assessment and concluded that, like the unphosphorylated native enzyme, Ca(2+) and vanadate exert a strong influence over the global structure of this low fluorescence phosphoenzyme. Based on a structural model with fluorescein isothiocyanate bound at the ATP site, we suggest that the stability as well as the low fluorescence of this phosphoenzyme is due to a fluorescein-mediated cross-link between two cytoplasmic domains that prevents hydrolysis of the aspartyl phosphate. Finally, we consider the alternative possibility that phosphate transfer to fluorescein itself could explain the properties of this low fluorescence species.