E2P Ground State Research Articles

Conformational changes during the reaction cycle of Plasma Membrane Ca2+-ATPase in the autoinhibited and activated states

Plasma membrane Ca2+-ATPase (PMCA) transports Ca2+ by a reaction cycle including phosphorylated intermediates. Calmodulin binding to the C-terminal tail disrupts autoinhibitory interactions, activating the pump. To assess the conformational changes during the reaction cycle, we studied the structure of different PMCA states using a fluorescent probe, hydrophobic photolabeling, controlled proteolysis and Ca2+-ATPase activity. Our results show that calmodulin binds to E2P-like states, and during dephosphorylation, the hydrophobicity in the nucleotide-binding pocket decreases and the Ca2+ binding site becomes inaccessible to the extracellular medium. Autoinhibitory interactions are disrupted in E1Ca and in the E2P ground state whereas they are stabilized in the E2·Pi product state. Finally, we propose a model that describes the conformational changes during the Ca2+ transport of PMCA.

Binding of cardiotonic steroids to Na+,K+-ATPase in the E2P state

The sodium pump (Na+, K+-ATPase, NKA) is vital for animal cells, as it actively maintains Na+ and K+ electrochemical gradients across the cell membrane. It is a target of cardiotonic steroids (CTSs) such as ouabain and digoxin. As CTSs are almost unique strong inhibitors specific to NKA, a wide range of derivatives has been developed for potential therapeutic use. Several crystal structures have been published for NKA-CTS complexes, but they fail to explain the largely different inhibitory properties of the various CTSs. For instance, although CTSs are thought to inhibit ATPase activity by binding to NKA in the E2P state, we do not know if large conformational changes accompany binding, as no crystal structure is available for the E2P state free of CTS. Here, we describe crystal structures of the BeF3- complex of NKA representing the E2P ground state and then eight crystal structures of seven CTSs, including rostafuroxin and istaroxime, two new members under clinical trials, in complex with NKA in the E2P state. The conformations of NKA are virtually identical in all complexes with and without CTSs, showing that CTSs bind to a preformed cavity in NKA. By comparing the inhibitory potency of the CTSs measured under four different conditions, we elucidate how different structural features of the CTSs result in different inhibitory properties. The crystal structures also explain K+-antagonism and suggest a route to isoform specific CTSs.

A kinetic comparison between E2P and the E2P-like state induced by a beryllium fluoride complex in the Na,K-ATPase. Interactions with Rb+

Metal-fluoride complexes have been used to induce E2P-like states with the aim of studying the events that occur during E2P hydrolysis in P-type ATPases. In the present work, we compared the E2P-like state induced by a beryllium fluoride complex (BeFx) with the actual E2P state formed through backdoor phosphorylation of the Na,K-ATPase. Formation of E2P and E2P-like states were investigated employing the styryl dye RH421. We found that BeFx is the only fluorinated phosphate analog that, like Pi, increases the RH421 fluorescence. The observed rate constant, kobs, for the formation of E2P decreases with [Pi] whereas that of E2BeFx increases with [BeFx]. This might wrongly be taken as evidence of a mechanism where the binding of BeFx induces a conformational transition. Here, we rather propose that, like for Pi, binding of BeFx follows a conformational-selection mechanism, i.e. it binds to the E2 conformer forming a complex that is much more stable than E2P, as seen from its impaired capacity to return to E1 upon addition of Na+. Although E2P and E2BeFx are able to form states with 2 occluded Rb+, both enzyme complexes differ in that the affinity for the binding and occlusion of the second Rb+ is much lower in E2BeFx than in E2P. The higher rates of Rb+ occlusion and deocclusion observed for E2BeFx, as compared to those observed for other E2P-like transition and product states suggest a more open access to the cation transport sites, supporting the idea that E2BeFx mimics the E2P ground state.

Crystal Structure of the Vanadate-Inhibited Ca2+-ATPase

Vanadate is the hallmark inhibitor of the P-type ATPase family; however, structural details of its inhibitory mechanism have remained unresolved. We have determined the crystal structure of sarcoplasmic reticulum Ca(2+)-ATPase with bound vanadate in the absence of Ca(2+). Vanadate is bound at the catalytic site as a planar VO3(-) in complex with water and Mg(2+) in a dephosphorylation transition-state-like conformation. Validating bound VO3(-) by anomalous difference Fourier maps using long-wavelength data we also identify a hitherto undescribed Cl(-) site near the dephosphorylation site. Crystallization was facilitated by trinitrophenyl (TNP)-derivatized nucleotides that bind with the TNP moiety occupying the binding pocket that normally accommodates the adenine of ATP, rationalizing their remarkably high affinity for E2P-like conformations of the Ca(2+)-ATPase. A comparison of the configurations of bound nucleotide analogs in the E2·VO3(-) structure with that in E2·BeF3(-) (E2P ground state analog) reveals multiple binding modes to the Ca(2+)-ATPase.

Quantifying bond distortions in transient enzyme species by a combination of density functional theory calculations and time-resolved infrared difference spectroscopy. Implications for the mechanism of dephosphorylation of the sarcoplasmic reticulum Ca2 +-ATPase (SERCA1a)

The sarcoplasmic Ca2+-ATPase (SERCA1a) forms two phosphoenzyme intermediates during Ca2+ pumping. The second intermediate E2P hydrolyzes rapidly, which is essential for the rapid removal of Ca2+ from the cytosol of muscle cells. The present work studies whether a weakening of the scissile PO bond in the E2P ground state facilitates dephosphorylation. To this end, the experimentally known vibrational spectrum of the E2P phosphate group was calculated with density functional theory (DFT) using structural models at two levels of structural complexity: (i) Models of acetyl phosphate in simple environments and (ii) ~150 atom models of the catalytic site. It was found that the enzyme environment distorts the structure of the phosphate group: one of the terminal PO bonds is shorter in the catalytic site indicating weaker interactions than in water. However, the bond that bridges phosphate and Asp351 is unaffected. This indicates that the scissile PO bond is not weakened by the enzyme environment of E2P. A second finding was that the catalytic site of the E2P state in aqueous solution appears to adopt a structure as in the crystals with BeF3−, where the ATPase is in a non-reactive conformation. The reactant state of the dephosphorylation reaction differs from the E2P ground state: Glu183 faces Asp351 and positions the attacking water molecule. This state has a 0.04Å longer, and thus weaker, bridging PO bond. The reactant state is not detected in our experiments, indicating that its energy is at least 1kcal/mol higher than that of the E2P ground state.

Critical Roles of Interdomain Interactions for Modulatory ATP Binding to Sarcoplasmic Reticulum Ca2+-ATPase

ATP has dual roles in the reaction cycle of sarcoplasmic reticulum Ca(2+)-ATPase. Upon binding to the Ca2E1 state, ATP phosphorylates the enzyme, and by binding to other conformational states in a non-phosphorylating modulatory mode ATP stimulates the dephosphorylation and other partial reaction steps of the cycle, thereby ensuring a high rate of Ca(2+) transport under physiological conditions. The present study elucidates the mechanism underlying the modulatory effect on dephosphorylation. In the intermediate states of dephosphorylation the A-domain residues Ser(186) and Asp(203) interact with Glu(439) (N-domain) and Arg(678) (P-domain), respectively. Single mutations to these residues abolish the stimulation of dephosphorylation by ATP. The double mutation swapping Asp(203) and Arg(678) rescues ATP stimulation, whereas this is not the case for the double mutation swapping Ser(186) and Glu(439). By taking advantage of the ability of wild type and mutant Ca(2+)-ATPases to form stable complexes with aluminum fluoride (E2·AlF) and beryllium fluoride (E2·BeF) as analogs of the E2·P phosphoryl transition state and E2P ground state, respectively, of the dephosphorylation reaction, the mutational effects on ATP binding to these intermediates are demonstrated. In the wild type Ca(2+)-ATPase, the ATP affinity of the E2·P phosphoryl transition state is higher than that of the E2P ground state, thus explaining the stimulation of dephosphorylation by nucleotide-induced transition state stabilization. We find that the Asp(203)-Arg(678) and Ser(186)-Glu(439) interdomain bonds are critical, because they tighten the interaction with ATP in the E2·P phosphoryl transition state. Moreover, ATP binding and the Ser(186)-Glu(439) bond are mutually exclusive in the E2P ground state.

A Structural View on the Functional Importance of the Sugar Moiety and Steroid Hydroxyls of Cardiotonic Steroids in Binding to Na,K-ATPase*

The Na,K-ATPase is specifically inhibited by cardiotonic steroids (CTSs) like digoxin and is of significant therapeutic value in the treatment of congestive heart failure and arrhythmia. Recently, new interest has arisen in developing Na,K-ATPase inhibitors as anticancer agents. In the present study, we compare the potency and rate of inhibition as well as the reactivation of enzyme activity following inhibition by various cardiac glycosides and their aglycones at different pH values using shark Na,K-ATPase stabilized in the E2MgPi or in the E2BeFx conformations. The effects of the number and nature of various sugar residues as well as changes in the positions of hydroxyl groups on the β-side of the steroid core of cardiotonic steroids were investigated by comparing various cardiac glycoside compounds like ouabain, digoxin, digitoxin, and gitoxin with their aglycones. The results confirm our previous hypothesis that CTS binds primarily to the E2-P ground state through an extracellular access channel and that binding of extracellular Na(+) ions to K(+) binding sites relieved the CTS inhibition. This reactivation depended on the presence or absence of the sugar moiety on the CTS, and a single sugar is enough to impede reactivation. Finally, increasing the number of hydroxyl groups of the steroid was sterically unfavorable and was found to decrease the inhibitory potency and to confer high pH sensitivity, depending on their position on the steroid β-face. The results are discussed with reference to the recent crystal structures of Na,K-ATPase in the unbound and ouabain-bound states.

Metal Fluoride Complexes of Na,K-ATPase: CHARACTERIZATION OF FLUORIDE-STABILIZED PHOSPHOENZYME ANALOGUES AND THEIR INTERACTION WITH CARDIOTONIC STEROIDS

The Na,K-ATPase belongs to the P-type ATPase family of primary active cation pumps. Metal fluorides like magnesium-, beryllium-, and aluminum fluoride act as phosphate analogues and inhibit P-type ATPases by interacting with the phosphorylation site, stabilizing conformations that are analogous to specific phosphoenzyme intermediates. Cardiotonic steroids like ouabain used in the treatment of congestive heart failure and arrhythmias specifically inhibit the Na,K-ATPase, and the detailed structure of the highly conserved binding site has recently been described by the crystal structure of the shark Na,K-ATPase in a state analogous to E2·2K(+)·P(i) with ouabain bound with apparently low affinity (1). In the present work inhibition, and subsequent reactivation by high Na(+), after treatment of shark Na,K-ATPase with various metal fluorides are characterized. Half-maximal inhibition of Na,K-ATPase activity by metal fluorides is in the micromolar range. The binding of cardiotonic steroids to the metal fluoride-stabilized enzyme forms was investigated using the fluorescent ouabain derivative 9-anthroyl ouabain and compared with binding to phosphorylated enzyme. The fastest binding was to the Be-fluoride stabilized enzyme suggesting a preformed ouabain binding cavity, in accord with results for Ca-ATPase where Be-fluoride stabilizes the E2-P ground state with an open luminal ion access pathway, which in Na,K-ATPase could be a passage for ouabain. The Be-fluoride stabilized enzyme conformation closely resembles the E2-P ground state according to proteinase K cleavage. Ouabain, but not its aglycone ouabagenin, prevented reactivation of this metal fluoride form by high Na(+) demonstrating the pivotal role of the sugar moiety in closing the extracellular cation pathway.

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

Modulatory ATP Binding Affinity in Intermediate States of E2P Dephosphorylation of Sarcoplasmic Reticulum Ca2+-ATPase

The mechanism of ATP modulation of E2P dephosphorylation of sarcoplasmic reticulum Ca(2+)-ATPase wild type and mutant forms was examined in nucleotide binding studies of states analogous to the various intermediates of the dephosphorylation reaction, obtained by binding of metal fluorides, vanadate, or thapsigargin. Wild type Ca(2+)-ATPase displays an ATP affinity of 4 μM for the E2P ground state analog, 1 μM for the E2P transition state and product state analogs, and 11 μM for the E2 dephosphoenzyme. Hence, ATP binding stabilizes the transition and product states relative to the ground state, thereby explaining the accelerating effect of ATP on dephosphorylation. Replacement of Phe(487) (N-domain) with serine, Arg(560) (N-domain) with leucine, or Arg(174) (A-domain) with alanine or glutamate reduces ATP affinity in all E2/E2P intermediate states. Alanine substitution of Ile(188) (A-domain) increases the ATP affinity, although ATP acceleration of dephosphorylation is disrupted, thus indicating that the critical role of Ile(188) in ATP modulation is mechanistically based rather than being associated with the binding of nucleotide. Mutants with alanine replacement of Lys(205) (A-domain) or Glu(439) (N-domain) exhibit an anomalous inhibition by ATP of E2P dephosphorylation, due to ATP binding increasing the stability of the E2P ground state relative to the transition state. The ATP affinity of Ca(2)E2P, stabilized by inserting four glycines in the A-M1 linker, is similar to that of the E2P ground state, but the Ca(2+)-free E1 state of this mutant exhibits 3 orders of magnitude reduction of ATP affinity.

Trinitrophenyl derivatives bind differently from parent adenine nucleotides to Ca 2+ -ATPase in the absence of Ca 2+

Trinitrophenyl derivatives of adenine nucleotides are widely used for probing ATP-binding sites. Here we describe crystal structures of Ca(2+)-ATPase, a representative P-type ATPase, in the absence of Ca(2+) with bound ATP, trinitrophenyl-ATP, -ADP, and -AMP at better than 2.4-Å resolution, stabilized with thapsigargin, a potent inhibitor. These crystal structures show that the binding mode of the trinitrophenyl derivatives is distinctly different from the parent adenine nucleotides. The adenine binding pocket in the nucleotide binding domain of Ca(2+)-ATPase is now occupied by the trinitrophenyl group, and the side chains of two arginines sandwich the adenine ring, accounting for the much higher affinities of the trinitrophenyl derivatives. Trinitrophenyl nucleotides exhibit a pronounced fluorescence in the E2P ground state but not in the other E2 states. Crystal structures of the E2P and E2 ∼ P analogues of Ca(2+)-ATPase with bound trinitrophenyl-AMP show that different arrangements of the three cytoplasmic domains alter the orientation and water accessibility of the trinitrophenyl group, explaining the origin of "superfluorescence." Thus, the crystal structures demonstrate that ATP and its derivatives are highly adaptable to a wide range of site topologies stabilized by a variety of interactions.

How processing of aspartylphosphate is coupled to lumenal gating of the ion pathway in the calcium pump

Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum is the best-studied member of the P-type or E1/E2 type ion transporting ATPases. It has been crystallized in seven different states that cover nearly the entire reaction cycle. Here we describe the structure of this ATPase complexed with phosphate analogs BeF(3)(-) and AlF(4)(-) in the absence of Ca(2+), which correspond to the E2P ground state and E2 approximately P transition state, respectively. The luminal gate is open with BeF(3)(-) and closed with AlF(4)(-). These and the E1 approximately P.ADP analog crystal structures show that a two-step rotation of the cytoplasmic A-domain opens and closes the luminal gate through the movements of the M1-M4 transmembrane helices. There are several conformational switches coupled to the rotation, and the one in the cytoplasmic part of M2 has critical importance. In the second step of rotation, positioning of one water molecule couples the hydrolysis of aspartylphosphate to closing of the gate.

Highlights from the Literature

Highlights from the Literature

Distinct Natures of Beryllium Fluoride-bound, Aluminum Fluoride-bound, and Magnesium Fluoride-bound Stable Analogues of an ADP-insensitive Phosphoenzyme Intermediate of Sarcoplasmic Reticulum Ca2+-ATPase: CHANGES IN CATALYTIC AND TRANSPORT SITES DURING PHOSPHOENZYME HYDROLYSIS

The structural natures of stable analogues for the ADP-insensitive phosphoenzyme (E2P) of Ca(2+)-ATPase formed in sarcoplasmic reticulum vesicles, i.e. the enzymes with bound beryllium fluoride (BeF.E2), bound aluminum fluoride (AlF.E2), and bound magnesium fluoride (MgF.E2), were explored and compared with those of actual E2P formed from P(i) without Ca(2+). Changes in trinitrophenyl-AMP fluorescence revealed that the catalytic site is strongly hydrophobic in BeF.E2 as in E2P but hydrophilic in MgF.E2 and AlF.E2; yet, the three cytoplasmic domains are compactly organized in these states. Thapsigargin, which was shown in the crystal structure to fix the transmembrane helices and, thus, the postulated Ca(2+) release pathway to lumen in a closed state, largely reduced the tryptophan fluorescence in BeF.E2 as in E2P, but only very slightly (hence, the release pathway is likely closed without thapsigargin) in MgF.E2 and AlF.E2 as in dephosphorylated enzyme. Consistently, the completely suppressed Ca(2+)-ATPase activity in BeF-treated vesicles was rapidly restored in the presence of ionophore A23187 but not in its absence by incubation with Ca(2+) (over several millimolar concentrations) at pH 6, and, therefore, lumenal Ca(2+) is accessible to reactivate the enzyme. In contrast, no or only very slow restoration was observed with vesicles treated with MgF and AlF even with A23187. BeF.E2 thus has the features very similar to those characteristic of the E2P ground state, although AlF.E2 and MgF.E2 most likely mimic the transition or product state for the E2P hydrolysis, during which the hydrophobic nature around the phosphorylation site is lost and the Ca(2+) release pathway is closed. The change in hydrophobic nature is probably associated with the change in phosphate geometry from the covalently bound tetrahedral ground state (BeF(3)(-)) to trigonal bipyramidal transition state (AlF(3) or AlF(4)(-)) and further to tetrahedral product state (MgF(4)(2-)), and such change likely rearranges transmembrane helices to prevent access and leakage of lumenal Ca(2+).