The Na+,K+-ATPase in complex with beryllium fluoride mimics an ATPase phosphorylated state

The Na+,K+-ATPase generates electrochemical gradients of Na+ and K+ across the plasma membrane via a functional cycle that includes various phosphoenzyme intermediates. However, the structure and function of these intermediates and how metal fluorides mimick them require further investigation. Here, we describe a 4.0 Å resolution crystal structure and functional properties of the pig kidney Na+,K+-ATPase stabilized by the inhibitor beryllium fluoride (denoted E2–BeFx). E2–BeFx is expected to mimic properties of the E2P phosphoenzyme, yet with unknown characteristics of ion and ligand binding. The structure resembles the E2P form obtained by phosphorylation from inorganic phosphate (Pi) and stabilized by cardiotonic steroids, including a low-affinity Mg2+ site near ion binding site II. Our anomalous Fourier analysis of the crystals soaked in Rb+ (a K+ congener) followed by a low-resolution rigid-body refinement (6.9–7.5 Å) revealed preocclusion transitions leading to activation of the dephosphorylation reaction. We show that the Mg2+ location indicates a site of initial K+ recognition and acceptance upon binding to the outward-open E2P state after Na+ release. Furthermore, using binding and activity studies, we find that the BeFx-inhibited enzyme is also able to bind ADP/ATP and Na+. These results relate the E2–BeFx complex to a transient K+- and ADP-sensitive E∗P intermediate of the functional cycle of the Na+,K+-ATPase, prior to E2P.

The flexibility and intermediary functional properties of the BeF x complex were anticipated earlier from the presence of a characteristic H + leak current mediated by Na + ,K + -ATPase (13) and in SERCA from the measurements of Ca 2+ occlusion (14) and Ca 2+ -mediated reactivation (15).
Here, we describe functional properties of the E2-BeF x complex of Na + ,K + -ATPase and structural rearrangements in its crystal structure induced by binding of Rb + as a K + congener. Close structural resemblance to the P i -induced E2P phosphoenzyme stabilized by cardiotonic steroids (10,11) includes outward-open ion-binding sites, which prompted investigations of the effect of extracellular Mg 2+ .
Notably, we also find that BeF x dissociation from the enzyme is accelerated by both nucleotide and ion bindingthese are characteristics that relate the E2-BeF x structure to the ADP-and K + -sensitive (E*P) phosphoenzyme.

Formation of the BeF x complex
The BeF x -inhibited complex of Na + ,K + -ATPase was formed by preincubation of the membraneous enzyme in 20 mM histidine (pH 7.0), 10 mM NaF, 0.5 mM MgCl 2 , 20 μM BeSO 4 , and used in all biochemical studies as well as initial material for crystallization.
Overall crystal structure of the BeF x complex of the Na + ,K + -ATPase The crystal structure of pig kidney Na + ,K + -ATPase (α 1 , β 1 , and FXYD2 also known as γ) was determined by molecular replacement (MR) using the ouabain-bound P i -induced (E2P-OBN) form as the starting model (Protein Data Bank [PDB] ID: 4HYT) (10), and at later stage compared with the structure of a different crystal form of the E2-BeF x complex (PDB ID: 7D91 (9)). The final model was refined against anisotropically truncated data extending to 4.05 Å, resulting in R work and R free values of 22.7% and 27.6%, respectively (Fig. 1A, Table 1).
BeF x coordinated to the conserved Asp369 phosphorylation site in the conserved DKTG segment of the P domain was identified in unbiased initial F o -F c difference map. Comparison shows similar arrangements of the P domains in both the P i -induced OBN-bound E2P state (10) and in the present BeF x structure. The TGES loop of the actuator (A) domain is in close proximity to BeF x and protects the phosphorylation site from spontaneous hydrolysis (Fig. 1A). High structural similarity with E2P-OBN (rmsd = 0.59 Å for all C α ), also reveal subtle OBN-induced rearrangements in the αM1-M4 segments of the earlier reported E2P-OBN structure. Slight , green (β-subunit), and hot pink (γ-subunit). Close-up view of the phosphorylation site is shown in the inset. Mg 2+ ions and the BeF x bound to Asp369 are depicted as green and teal spheres, respectively. The Post-Albers reaction scheme of Na + ,K + -ATPase accumulating 3-pool model of phosphoenzymes is shown in the upper right. B, overall comparison of E2-BeF x (blue) and ouabain-bound P i -induced E2P (pink). Mg 2+ ions are depicted as green spheres, and ouabain is colored in hot pink. The structures were aligned on the TM segment αM7-10. C, coordination of Mg 2+ by Glu327 (M4), Asn776, Glu779 (M5), and Asp804 (M6) side chains. Mg 2+ and water are depicted as green and red spheres, respectively. Density map contoured at 1.5 σ. D, extracellular access channel of Na + ,K + -ATPase E2-BeF x (blue) aligned with SERCA E2-BeF x (PDB ID: 3B9B (15), wheat). The structures were aligned by αM5-M6. Bound Mg 2+ ions are shown as spheres also in blue and wheat. E and F, water cavity representation of Na + ,K + -ATPase E2-BeF x (red) and SERCA E2-BeF x (purple), showing a narrower entrance to the ion-binding sites in the Na + ,K + -ATPase E2-BeF x complex. Cavities were calculated in HOLLOW (49). PDB, Protein Data bank; SERCA, sarco/ endoplasmic reticulum Ca 2+ -ATPase. outward tilting of the αM1-M2 bundle is due to the extensive hydrogen-bonding network between the β-surface of OBN and polar side chains of αM1, αM2, and αM6 (10). Furthermore, the inhibitor-bound site makes the extracellular part of the αM3-M4 helices close in, causing a 4 tilt of αM4 with pivot at Val322 (Fig. 1B) as compared with E2-BeF x .
The extracellular ion pathway is formed by the transmembrane (TM) αM1-M6 helices, which define an outwardopen arrangement similar to SERCA1a in the Mg 2+ -stabilized E2-BeF x form (15) (Fig. 1D). The arrangement of αM2-M5 helices in Na + ,K + -ATPase, however, is slightly more compact, and the residues lining the pathway have larger hydrophobic side chains (e.g., Tyr308, Phe316, Phe783, and Phe786 in Na + ,K + -ATPase corresponding to Phe279, Ile298, Ile775, and Thr778 in SERCA) (Fig. 1, E and F). The extracellular ion pathway is thus narrow but solvated, consistent with a low voltage sensitivity of K + binding through such a pathway (16) and a voltage-insensitive release of the third and last Na + ion from the Na + -bound state (17).
For the cation-binding sites in the TM domain, we observed a residual density near site II in the initial unbiased F o -F c difference map, similar to a Mg 2+ site in the E2P-OBN complex (10). Indeed, the E2-BeF x crystals were grown in the presence of 175 mM MgCl 2 , and Mg 2+ binding is likely to stabilize an open conformation as seen also for the E2-BeF x complex of SERCA1a (15), although the site is shifted (Fig. 1D). The Mg 2+ site seems to overlap with incoming K + (Fig. 1C) (see further).

P i -induced E2P and E2-BeF x complexes have similar functional properties
Structural similarities between P i -induced E2P and E2-BeF x complexes (Fig. 1B) are also supported by their functional properties, for example, by interactions with cardiotonic steroids. Figure 2 summarizes kinetics of anthroylouabain binding to these complexes, which, judged from the crystallographic data, have very similar organization of the CTS-binding cavities. The main conclusion from the kinetic experiments is that affinities of both forms to anthroylouabain are very high, with only small numeric differences in the values of association and dissociation rate constants. Although the effect of K + on anthroylouabain affinity seems to be less pronounced for E2-BeF x , it is still in the direction of decreased affinity for both forms.
Interactions of Na + , K + /Rb + , and Mg 2+ with the E2-BeF x complex The E2-BeF x crystal structure reveals that the cationbinding cavity is open to the extracellular side and occupied by a Mg 2+ ion. The homologous complex of SERCA in crystallized form also contained Mg 2+ (15) (Fig. 1F), whereas in functional studies, it was shown to bind and occlude Ca 2+ (14). Do the ions bind to the E2-BeF x complex of Na + ,K + -ATPase, and what are the functional consequences of this binding? We investigated the interactions with Na + , K + (Rb + as congener), and Mg 2+ . Values in parentheses are for the highest resolution shell.
where n h is the multiplicity, I hi is the ith intensity of reflection h, and CI h D is the weighted average intensity for all observations i of reflection h. c The Wilson plot had a linear appearance in the 4 to 5 Å resolution range, from which a Wilson B was derived. For the low-resolution datasets, the Wilson B is not very meaningful.
Mg 2+ binding does not affect Na + and K + binding The effect of extracelluar Mg 2+ on binding of Na + , K + , and H + was investigated by two-electrode voltage clamping on Xenopus laevis oocytes (a well-established model to study functional properties of the Na + ,K + -ATPase), expressing human α 2 β 1 (see sequence alignment, Fig. S1). In the absence of extracellular K + , the pump is distributed between the whole range of phosphoenzymes because of voltage-dependent binding and release of Na + . We assayed Na + binding in the presence of 0, 1, and 5 mM Mg 2+ and found no difference in either apparent affinity for Na + (Q/Vm curves) or the rate constants of release (Fig. S2, A and B). We next examined the potency and efficacy of K + binding by measuring the steadystate current at −30 mV in the presence of 115 mM Na + with or without 5 mM Mg 2+ at varying K + concentrations. Again, there were no differences in currents suggesting that physiological concentrations of extracellular Mg 2+ have no significant effects on binding of Na + or K + from the extracellular environment (Fig. S2, C and D).
However, in the absence of extracellular Na + and K + , an inwardly rectifying H + leak current is observed for the Na + ,K + -ATPase (7), in particular at hyperpolarized potentials. This inward leak, however, was significantly inhibited at hyperpolarized potentials by 5 mM or 20 mM Mg 2+ (Fig. 3).
Thus, the affinity for the extracellular Mg 2+ is so low that it does not interfere with Na + or K + binding under physiological conditions. Yet, when bound, it stabilizes the outward-open state and interferes with a H + leak current.

Beryllium fluoride prevents oligomycin-induced Na + occlusion
The E2-BeF x form of SERCA1a is capable of Ca 2+ occlusion (14). We therefore investigated Na + occlusion by the E2-BeF x form of the Na + ,K + -ATPase but failed to reveal any bound Na + (Fig. 4A). We repeated the experiment in the presence of oligomycin, which decreases the rate of Na + release (18,19) and found an expected increase in 22 Na + bound to the E1 state, where the Na + ,K + -ATPase occluded approximately 2.5 nmol per nmol protein (Fig. 4B). However, under the conditions of BeF x complex formation, the 22 Na + / 3 H + ratio was the same as in the absence of enzyme or oligomycin (Fig. 4A), implying that binding of BeF x effectively prevented Na + occlusion by the enzyme.

K + occlusion by the BeF x complex
BeF x binding to Na + ,K + -ATPase is associated with an increase of RH421 fluorescence, similar to the response of this dye to enzyme phosphorylation, whereas addition of K + ions  Structure-function of the E2-BeF x form of Na + ,K + -ATPase induces a decrease in fluorescence (Fig. 5A, inset, and (20)). The values for both amplitude and k obs for K-response extracted by fitting with a monoexponential function show a hyperbolic concentration dependence for the amplitude of fluorescence that decrease from its maximal level already at 2 mM KCl and a linear increase for k obs (Fig. 5). Knowing that K + interacts with P i -induced E2P in the same way (21), we assumed that direct binding of K + is followed by its occlusion in E2-BeF x form. Indeed, direct measurements with 86 Rb + (as K + congener) showed its accumulation in the E2-BeF x form (20).

Rb + binding and extracellular gate closure
Comparison of the E2-BeF x (this study) and [Rb 2 ]E2-MgF x (6) states reveals the overall transitions of the extracellular gate closure upon K + /Rb + binding. We tracked the binding process and mechanism of occlusion by crystal soaking procedures.

Preoccluded (Rb)E2-BeF x states
Structural rearrangements in the BeF x complex because of Rb + binding and occlusion were followed by soaking E2-BeF x crystals. Two datasets were collected at 6.9 and 7.5 Å maximum resolution (consisting of 11,991 and 9616 unique reflections, respectively) at a wavelength of 0.814 Å with a strong anomalous signal for Rb + . They represent transition intermediates from the native E2-BeF x with bound Mg 2+ (Fig. 6A) toward the earlier reported occluded structure of [Rb 2 ]E2-MgF x (PDB ID: 3KDP (6)). Indeed, the datasets for Rb + -soaked E2-BeF x (here referred to as quick soak and long soak corresponding to 10 mM, 20 s and 50 mM, 3 h, respectively) produced strong anomalous difference peaks (5.8-8.4 σ) at the cation-binding sites, reflecting exchange of Mg 2+ with Rb + (Fig. 6, B-D). As the asymmetric unit of the P2 1 2 1 2 1 crystals consists of two protomers, we have altogether four representations of (2) no protein, nonspecific binding after washing of the filter; (3) Na + ,K + -ATPase in the E2-BeF x form, followed by washing of the filter. The ratio was not changed by washing, indicating that both isotopes interact with filter in the same way. It was not affected by the presence of BeF x complex of the Na + ,K + -ATPase. B, in the presence of oligomycin. (1) Na + ,K + -ATPase in E1 conformation, followed by washing of the filter; (2) Na + ,K + -ATPase in the E2-BeF x form, followed by washing of the filter. The data from individual experiments as well as mean values ± SD are shown.  Quantitative determination of Rb + occupancy was challenging because of the low resolution of the datasets, but occupancy analyses through molecular replacement with single-wavelength anomalous diffraction refinement (MR-SAD, see Experimental procedures section) appeared consistent with two fully occupied binding sites for both protomers in the long Rb + soak (occupancy > 0.75). For the two quick soak conformations, one protomer was fully occupied, whereas the other showed partial occupancy (with an occupancy less than 0.1 for a second site, assuming a full occupancy of a first site). Interestingly, the anomalous difference peak for the partially occupied quick soak protomer overlapped with the Mg 2+ site between the two K + sites I and II, suggesting that initial Rb + /K + binding takes place at this exposed site prior to formation of the properly coordinated K + (or Rb + ) sites (Fig. 6B). That said, the low resolution obviously precludes firm conclusions on this point, but it should be noted that many thousand reflections contribute to the derived values. Rigid-body model refinement for the cytosolic domains and individual TM segments produced large improvements in crystallographic R-factors for model representations and converged robustly at conformational changes that were also consistent with unbiased omit map controls.
For all four protomers of the soaked crystals, the cytoplasmic domains exhibited weak density in the electron density maps, likely indicating flexibility (Figs. S5 and S6). The E2-BeF x MR model provided the relative position of the cytosolic headpiece based on some defined helices in each domain; thus, they are still modeled despite the weak density. The TM helices, however, were clearly visible in the electron density map to model the C α main chain. The two protomers in the quick soak are similar to each other (rmsd = 1.16 Å, all C α ; Fig. S5) and also show resemblance to protomer 1 in the long-soaked crystal (rmsd: 1.27 Å, all C α ). However, the last protomer (long soak protomer 2) has a different conformation for the TM helices (rmsd = 1.96 Å between C α of the two long soak protomers; Fig. S6). For simplicity, the protomers will therefore in the following be referred to as the initial (Rb)E2-BeF x binding form (quick soak conformations), the early (Rb 2 )E2-BeF x binding form (long soak protomer 1), and the late (Rb 2 ) E2-BeF x binding form (long soak protomer 2) when discussing structural changes.

Sequence of events
The refined atomic models for the E2-BeF x form (this study) and [Rb 2 ]E2-MgF x form (6) represent the start and end points of the K + binding, and the rigid body refined models of the soaked crystals, albeit determined at low resolution, provide valuable insights into the trajectories of the K + /Rb +induced conformational changes that activate the dephosphorylation reaction.
Superposition of the Rb + -soaked structures and the native E2-BeF x form based on the TM αM7-M10 segment revealed sequential closing movements of the M1-M4 helices (Fig. 7). K + binding associates the extracellular part of αM4 to site II, most likely through main chain carbonyls of Val322, Ala323, and Val325 engaging in coordination (6,22). In the initial binding (Rb)E2-BeF x form, this results in αM4 tilting 7 toward αM6 (Fig. 7, A and B). In the early (Rb 2 )E2-BeF x form, αM4 is further tilted 5 , and the extracellular gate is closing. Through van der Waals contacts between αM1, αM2, and αM4, the αM1-M2 segment follows αM4 to an intermediate position via a 2 Å translation toward the extracellular side (Fig. 7, C and D).
In the next step, reaching the late (Rb 2 )E2-BeF x form, the αM1-M2 helices are further translated toward to extracellular side (4.5 Å) and 6 lateral tilted toward the cytosolic side. The cytoplasmic part of the αM2 helix bends toward the A-αM3 linker region (Fig. 7, E and F). The change of path is likely realized by a partial unwinding of the αM2 helix (M2 switch (23)), which also gains flexibility; as is indicated by poor density for the cytoplasmic end of αM2 (Fig. S6). The unwinding enables the A domain to rotate 7 around the phosphorylation site of the P domain (toward the membrane). To complete the transition to the fully occluded [Rb 2 ]E2-MgF x complex (PDB ID: 3KDP, (6)), the A domain must finish its rotation (7 ), bringing the TGES motif into dephosphorylation mode. This rotation causes a further 1.5 Å translation of the αM1-M2 segment toward the cytoplasmic side and a further unwinding of the αM2 cytoplasmic end between Glu144 and Ile150. Stabilizing the fully occluded state, the segment Ile150-Lys155 rewinds to form a hydrophobic cluster (24,25) that ensures tight association between αM2-A and the A-αM3 linker segment with the P domain (Fig. 7, G and H).

Changes in the α-C-terminal region and β-subunit
The α-C-terminal region plays an important role in the transport cycle (6,31,32). Mutations in the region are associated with neurological disorders, and the C terminus seems integral to the function of the Na + site III and protonation of K + -bound states (31). However, when comparing the E2-BeF x and [Rb 2 ]E2-MgF x structures in a local αM8 helix superimposition, only subtle changes are seen. Although they may affect the position and dynamics of the conserved C-terminal tyrosine residue (Fig. S9A) and thereby solvent access and protonation of the Na + site III, functionally important features are difficult to qualify from the current study. Similarly, the β-subunit TM helix (β-TM) makes a small lateral shift toward αM7 (2 ) at Rb + occlusion.
On the other hand, the flexible N-terminal tail of the β-subunit (Phe15-Ser31 modeled) undergoes a different twist in the Rb + -occluded state, changing its interaction with the α-subunit. In the E2-BeF x state, βArg27 likely forms a salt bridge with Glu1013 (αM10 0 ), which gets disrupted in the Rb +occluded state, where βArg27 is instead exposed to the cytosol. The N-terminal tail of the β-subunit appears more extended, as βAsn18 (the resolved β-N terminus of [Rb 2 ]E2-MgF x ) is shifted 6.5 Å compared with E2-BeF x , in response to changes Structure-function of the E2-BeF x form of Na + ,K + -ATPase in position of the cytoplasmic domains of the α-subunit in the E2P-E2⋅P i transition (Fig. S9B).

Binding of ions and nucleotides induces dissociation of BeF x from the Na + ,K + -ATPase
Monitoring the recovery of Na + ,K + -ATPase activity, we investigated the effect of ions and nucleotides on the E2-BeF x complex (Fig. 8). The time course of the reactivation of ATPase activity reflects the rate of BeF x dissociation from the enzyme. Spontaneous dissociation in the media with 100 mM NMG + (included to compensate for possible effects of ionic strength) is slow but increases in the presence of specific ions (Fig. 8A). K + seems to be more efficient than Na + , as would be expected for extracellular ion binding. Note that Na + concentration in the experiment is twofold higher than that of K + , whereas total ion concentrations are always 100 mM, adjusted with NMG + .
Also ADP (and ATP, but ADP formation during preincubation with ATP cannot be avoided) speeded up reactivation of the ATPase activity compared with NMG + alone, and this effect is further amplified by Na + . On the other hand, K + completely cancels the ADP effect, presumably by preventing its binding by allosterism.
Interestingly, the rate of dephosphorylation of the P iinduced E2P form does not increase upon addition of neither ADP nor K + (21,33), so our data point to an apparently significant difference between the two otherwise analogous conformations (P i -induced E2P and E2-BeF x ). The explanation is, however, relatively straightforward: high stability of the E2-BeF x form allows both ions and nucleotides to equilibrate with the protein and to influence and stimulate BeF x dissociation. The acyl-phosphate bond on the other hand is reactive, and P i -induced E2P dephosphorylates with the rate constant of approximately 60 min −1 (21), that is, faster than equilibration with other ligands.

Docking of ADP into E2-BeF x structure
Visualization of ADP interaction with E2-BeF x was approached by docking the ADP molecule from the [Na 3 ]E1-AlF x -ADP structure (PDB ID: 3WGV (34)) into the E2-BeF x state guided by a local structural alignment of the N domains. ADP fits well into the N domain of the E2-BeF x complex (with Asp443 and Glu446 within coordination distance) and requires only minor rearrangement for the Phe475-Gln482 loop (Fig. 9A). The A domain comes into close contact with Lys205 near the β-phosphate of ADP (<1.9 Å), which presumably will cause a slight repositioning of the A domain. In the E2-MgF x state, the interface between the N and A domains is only stabilized by an ionic bond between Arg544 (N domain) and Glu216 (A domain). Thus, it is likely that Arg544 either interacts with the β-phosphate of ADP upon its binding to E2-BeF x (and allows further interaction with Na + ) or forms a salt bridge with Glu216 as consequence of interactions with K + . Both destabilizing effects are likely to provide greater mobility of the A domain and accelerate BeF x dissociation.

Discussion
Comparison of the functional properties of the P i -induced E2P and E2-BeF x revealed close similarities of these forms: kinetics of interactions with the cardiotonic steroids and K + are virtually the same, and the structures of E2-BeF x and E2P stabilized by cardiotonic steroids show clear structural resemblance.
The cation-binding sites are open to the extracellular side and accept Na + , K + , as well as Mg 2+ at low affinity. The ability of E2-BeF x to occlude Na + is lost, even in the presence of oligomycin, and Na + affinity is therefore low. Na + binding, however, destabilizes E2-BeF x , in a way analogous to the dephosphorylation under the Na + /Na + exchange reaction associated with ATP hydrolysis (35).
Binding of K + (or the congener Rb + ) triggers the conformational rearrangements necessary for ion occlusion and destabilization of the Asp369-BeF x bond. Rb + soaking of the E2-BeF x crystals revealed a sequence of Rb + preocclusion steps. Although only qualitative, the occupancy analysis revealed that the initial (Rb)E2-BeF x binding form has only partial occupation at site I. Furthermore, the anomalous difference suggests an overlap of the initial site with the Mg 2+ site (Fig. 6B). This indicates that going from the E2-BeF x open state, K + will bind in a sequential manner by first binding to an initial site that likely overlaps with the low-affinity Mg 2+ site coordinated by Asn776, Glu779 (αM5), and Asp804 (αM6) (10). Subsequently, the initial site changes configuration to the Figure 8. Restoration of the Na + ,K + -ATPase activity due to dissociation of BeF x . (A) Time course of BeF x dissociation from the Na + ,K + -ATPase induced by cations. Note, that the Na + concentration is twofold higher than that of K + . Total ion concentration is always 100 mM, adjusted with NMG + when necessary (B). Time course of BeF x dissociation induced by nucleotides and cations.
Structure-function of the E2-BeF x form of Na + ,K + -ATPase K + site I, at the same time making space for ion binding at site II. That involves main-chain carbonyls of αM4 and ultimately leads to closure of the extracellular gate. This mechanism, described from the outward-open E2-BeF x form and onward, is consistent with exchange kinetics followed in the opposite direction. Thus, in the occluded [K2]E2-MgF x form, the exchange with extracellular ions occurs first at site II (36). The oscillations of αM3-M4 helices allowing only one ion to bind at the time stand for the "flickering gate," first proposed by Forbush (37) on the basis of deocclusion experiments and later supported by high-speed voltage-clamp measurements (16).
In summary, the conformational transitions observed in soaking experiments are the prelude to the dephosphorylation process in the Na,K-ATPase reaction. The sequential binding of two K + through the αM1-M2-triggered rotation of the A domain rearranges the TGES motif, allowing water access and desphosphorylation (Fig. S10).
The aforementioned interactions with K + suggest that the E2-BeF x complex is a mimic of the ground state E2P. However, E2-BeF x is also destabilized by ADP, that is, it has a property of an ADP-sensitive state (Fig. 8B). The ability of ADP to induce a direct hydrolysis of the acyl-phosphate bond was described earlier by Hobbs et al. (4) for the phosphoenzyme E*P. ADP-induced dephosphorylation of E*P occurred directly, without transition to E1P. In the case of E2-BeF x , however, a transition toward E1-BeF x might be induced by the simultaneous presence of ADP and Na + , since the synergy between these two ligands is very clear (Fig. 8B). K + , on the other hand, cancels the ADP effect (Fig. 8B). Because of occlusion, it prohibits ADP binding and leads to slow BeF x dissociation from the [K 2 ]E2-BeF x complex.
In conclusion, the E2-BeF x complex of Na + ,K + -ATPase represents a phosphorylated intermediate that can reach both E2P and E*P. The structural similarity to the backdoor phosphorylated (P i -induced) E2P form is obvious from its comparison with the CTS-stabilized complexes of the Na,K-ATPase. Functionally, P i -induced E2P and E2-BeF x react nearly identically with CTS (here demonstrated by anthroylouabain interactions) and with K + (probed by RH421 fluorescence), and both forms are capable of Rb + or K + occlusion. Soaking of the E2-BeF x crystals with Rb + allowed visualization of different steps in the occlusion process. At the same time, firm coordination of BeF x within the phosphorylation site grants time for nucleotide binding and exploration of a dynamic E*P intermediate, unlike for E2P. These studies should be very informative for future investigations of P-type ATPase dynamics and studies of, for example, mutant forms.

Protein preparation
Pig kidney Na + ,K + -ATPase was purified as previously described (38). The specific ATPase activity of the Na + ,K + -ATPase-purified membrane preparations was about 1800 μmol P i per hour per mg of membrane protein at 37 C.

Crystallization and data collection
The BeF x -inhibited complex of Na + ,K + -ATPase was formed by preincubation of the membraneous enzyme in 20 mM histidine (pH 7.0), 10 mM NaF, 0.5 mM MgCl 2 , and 20 μM BeSO 4 . The stabilized E2-BeF x complex was subsequently solubilized in the same buffer with the nonionic detergent octaethyleneglycol mono-n-dodecylether [C 12 E 8 ] at a ratio of 0.9 mg C 12 E 8 per mg protein, and insoluble material was removed by ultracentrifugation. The final concentration of solubilized protein was 9 to 10 mg/ml. Crystals were grown by vapor diffusion at 15 C in 2 μl hanging drops for 2 to 3 weeks. The protein sample was mixed in a 1:1 ratio with reservoir solution containing 16.5% (w/v) polyethylene glycol 2000 monomethyl ether, 10% (v/v) glycerol, 175 mM MgCl 2 , 150 mM NaCl, 20 mM Hepes/Mes (pH 6.2) and 2 mM DTT. The crystals were dehydrated overnight at 4 C against a 30% polyethylene glycol 2000 monomethyl ether reservoir solution before flash cooling. The final datasets were collected at 100 K on the DESY-EMBL beamline P14 and the SLS-X06DA (PXIII) beamline. For Rb + soaks, 1.1 mM sucrose monodecanoate was added to the solubilized protein before mixing with reservoir solution. About 10 and 50 mM RbCl, respectively, was added to the crystallization drop and equilibrated for 20 s and 3 h, respectively. The final datasets of Rb + -soaked crystals were collected on the DESY-EMBL beamline P13 and the DLS-I24 beamline using a wavelength of 0.814 Å.

Data processing
Datasets were processed and scaled using XDS software (Wolfgang Kabsch, MPI for Medical Research) (39). The crystals showed P2 1 2 1 2 1 space group symmetry with unit Structure-function of the E2-BeF x form of Na + ,K + -ATPase cell dimensions, a = 116.3 Å, b = 117.8 Å, and c = 490.9 Å and two αβγ heterotrimers per asymmetric unit. For the native E2-BeF x structure, datasets derived from two crystals were merged to yield the final dataset. The data were anisotropically scaled using the Diffraction Anisotropy Server (http://services.mbi. ucla.edu/anisoscale) (40), setting the resolution limits to 5.4, 4.4, and 4.0 Å along a*, b*, and c*, respectively. MR was performed using PHASER (Phenix supported program) (41). As a search model, we used the crystal structure of Na + ,K + -ATPase E2P OBN complex (PDB ID: 4HYT (10)) and later also the structure of a different crystal form of the E2-BeF x complex (PDB ID: 7D91) (9). Rigid body refinement followed by simulated annealing refinement protocol was performed in PHENIX (Phenix Industrial Consortium) (42). Manual refinement was carried out in Coot (MRC-LMB) (43), and the further model refinement was continued in PHENIX using noncrystallographic symmetry (NCS), translation-librationscrew parameterization, and grouped atomic displacement parameter refinement. Because of low resolution of the data, tight geometry restraints were imposed on the model to stabilize the refinement. Rigid body groups were defined by the A, N, and P domains along with the αM1-2, αM3-4, αM5-10/ βM/γM, and β-ectodomain. NCS and translation-librationscrew groups were defined by the A, N, and P domains, the TM domain αM1-10/βM/γM, and the β-ectodomain. The quality of the final model was assessed using the MOLPRO-BITY server (Duke Biochemistry, Duke University School of Medicine) (44). For Rb + -soaked datasets, the diffraction data were first scaled using ScaleIT (CCP4 supported program) (45) to the Na + ,K + -ATPase E2P OBN complex (PDB ID: 4HYT) (10), and the initial phases were obtained by MR using PHASER and the E2-BeF x structure. Rigid body refinement, NCS, and atomic displacement parameter refinement (same groups as for the E2-BeF x structure) were performed for each dataset. Occupancy calculations were performed using MR with single-wavelength anomalous diffraction (46). All protein structure figures were prepared using PyMOL (The PyMOL Molecular Graphics System, version 2.3.0 [Schrödinger LLC, 2012]).

Na + occlusion
The membrane-bound enzyme (0.5 mg/ml) was incubated with 1 mM 22 NaF in the presence of 10 mM Tris-HCl (pH 7.0), 0.5 mM MgCl 2 , 1 mM 3 H-glucose in the presence or the absence of 0.02 mM BeSO 4 , with or without 0.02 mg/ml oligomycin for 5 h at 0 C. Then, 0.4 ml of the mixture was loaded on a Millipore HAWP 0.45 μm filter and immediately washed with 1 ml of Tris-HCl buffer containing 20 mM NaCl to decrease background. The filters were counted in 4 ml Packard Filtercount scintillation fluid. 3 H-counts gave an estimate for the nonspecific absorption of the isotopes of the filter and for the amount trapped in the wetting volume.
Dissociation of BeF x from the Na + ,K + -ATPase induced by different ligands BeF x complex of Na + ,K + -ATPase was formed by preincubation of the enzyme in 20 mM histidine (pH 7.0), 10 mM NaF, 0.5 mM MgCl 2 , and 20 μM BeSO 4 on ice overnight. The suspension was subjected to centrifugation 30 min × 50,000g at 4 C, and the pellet was resuspended in the media containing 20 mM histidine (pH 7.0) and varying ligands in concentrations as noted in Figure 8. After different periods of incubation at 37 C, the aliquots from each sample were used for measurement of the Na + ,K + -ATPase activity under optimal conditions (47).

RH421 fluorescence experiments
Equilibrium and transient RH421 fluorescence in response to varying concentrations of KCl was measured at room temperature in 20 mM histidine (pH 7.0), 10 mM NaF, 0.5 mM MgCl 2 , 0.05 mg/ml enzyme, and 0.2 μg/ml RH421 (VWR). Formation of BeF x complex was ensured by addition of 20 μM BeSO 4 . Measurements under equilibrium conditions were performed on a SPEX Fluorolog fluorometer in a cuvette with 1 cm light path with continuous stirring. Excitation wavelength was 580 nm (slit 2 nm) and emission 630 nm (slit 14 nm). Observed rate constants were obtained in the experiments using rapid-mixing stopped-flow spectrofluorometer (Applied Photophysics) at excitation wavelength of 580 nm (slit 1 nm) with 630 nm cutoff filter on the emission side.

Anthroylouabain fluorescence experiments
Equilibrium and transient experiments with anthroylouabain were performed at room temperature in either 20 mM histidine (pH 7.0), 4 mM H 3 PO 4 (adjusted with N-methyl-D-glutamine), 4 mM MgCl 2 , 0.05 mg/ml enzyme (P i -induced E2P complex); or 20 mM histidine (pH 7.0), 10 mM NaF, 0.5 mM MgCl 2 , 20 μM BeSO 4 , and 0.05 mg/ml enzyme (BeF x complex). When necessary, K + was added as KCl in the experiments with P iinduced E2P complex form, whereas in the case of E2-BeF x complex, 10 mM NaF was replaced by equimolar KF. A SPEX Fluorolog fluorometer was used to monitor slow reactions, excitation at 370 nm (slit 5 nm), emission at 485 nm (slit 5 nm). Association rate constants were obtained in the experiments using rapid-mixing stopped-flow spectrofluorometer at excitation wavelength of 370 nm (slit 1 nm) with 485 nm cutoff filter on the emission side.
Dissociation of anthroylouabain complexes was performed in a chase experiment as follows: the enzyme complexes were preincubated with 0.2 μM anthroylouabain overnight. Then 3.3 mM ouabain was added to the sample (anthroylouabain was diluted to 0.13 μM simultaneously), and decrease in fluorescence was monitored in time.

Two-electrode voltage clamping
The experiments were performed as described (48). In brief, mRNA was made with the mMESSAGE mMACHINE T7 Ultra Kit (Life Technologies) from linearized DNA encoding the human α2 with mutations to decrease OBN sensitivity (Q116R and N127D) and β1. X. laevis oocytes (EcoCyte Bioscience) were injected with 9.5/2.5 ng α2/β1 subunit mRNA in 50.6 nl. Oocytes were incubated 2 to 8 days at 12 to 18 C in ND96 buffer supplemented with 25 μg/ml gentamicin and 2.5 mM sodium pyruvate. Oocytes were clamped with an OC-725C amplifier (Warner Instruments), and the signal was digitized by a 1440A digitizer (Molecular Devices). Data were recorded with PClamp 10.4 (Molecular Devices) and analyzed with ClampFit (Molecular Devices) and GraphPad Prism (GraphPad Software, Inc). For sodium/sodium exchange recordings, the extracellular buffer contained 115 mM NaOH, 110 mM sulfamic acid, 10 mM Hepes, 5 mM BaCl 2 , 1 mM MgCl 2 , 0.5 mM CaCl 2 , 10 μM OBN, pH 7.4 (adjusted with sulfamic acid). For other recordings, NaOH was replaced by NMDG or KOH as indicated. Voltage jumps were performed from a holding potential of −50 mV with 200 ms jumps to potentials between 60 mV and −160 mV in 20 mV steps. Charge (Q) and rate currents were determined by subtracting a recording with 10 mM OBN added from the immediately preceding recording. On-and off-currents were fitted with single exponentials to determine rate currents and charge, respectively. Charges were fitted to a Boltzmann distribution. For evaluation of steady-state currents, the voltage protocol was performed in buffer, buffer with 10 mM OBN, and buffer. The last recording ensures stability throughout the recordings.

Data availability
Coordinates and diffraction data for the E2-BeF x structure and for the low-resolution models of the initial, early, and late stages of Rb + binding have been deposited in the PDB under accession codes 7QTV, 7YZR, and 7Z04.