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

Abstract

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-BeF_x). E2-BeF_x 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 (P_i) 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 BeF_x-inhibited enzyme is also able to bind ADP/ATP and Na⁺. These results relate the E2-BeF_x complex to a transient K⁺- and ADP-sensitive E∗P intermediate of the functional cycle of the Na⁺,K⁺-ATPase, prior to E2P.

Keywords: E1–E2; Na(+),K(+)-ATPase; P-type ATPase; beryllium fluoride; crystallography; inward-outward; membrane protein; transporter.

Figure 1

Crystal structure of E2–BeF_xstate of Na⁺,K⁺-ATPase.A, cartoon representation of E2–BeF_x state colored according to the different domains: red (nucleotide-binding [N] domain), blue (phosphorylation [P] domain), yellow (activator [A] domain), wheat (transmembrane [TM] domain, αM1–M10), green (β-subunit), and hot pink (γ-subunit). Close-up view of the phosphorylation site is shown in the inset. Mg²⁺ 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). Mg²⁺ 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 Mg²⁺ by Glu327 (M4), Asn776, Glu779 (M5), and Asp804 (M6) side chains. Mg²⁺ 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 Mg²⁺ 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 Ca²⁺-ATPase.

Figure 2

Interaction of P_i-induced E2P and BeF_xcomplexes of Na⁺,K⁺-ATPase with anthroylouabain. Data obtained with P_i-induced E2P are shown in black color, with BeF_x complex in red. A, changes in anthroylouabain (AO) fluorescence at two different concentrations, 0.25 μM (full line) and 0.75 μM (stippled line), were induced by its binding to the E2P and BeF_x complexes. Calculated second-order association rate constants for the reactions are shown in the inset. B, amplitude of the fluorescence change as function of AO concentration in the absence or the presence of 10 mM KCl. C, time course of AO dissociation from E2P and BeF_x complexes.

Figure 3

Inward leak current is affected by magnesium.A, the ouabain-sensitive steady-state current measured in the absence of extracellular sodium and potassium in response to membrane potential for different concentrations of magnesium. Current is normalized to −1 for 0 mM Mg²⁺ at −140 mV (n = 4). B, leak currents at −140 mV. For 5 and 20 mM, the currents are significantly smaller. Line at mean with SD (∗p < 0.05; ∗∗p < 0.0001; Student’s t test).

Figure 4

BeF_xbinding prevents Na⁺occlusion in the presence of oligomycin. Counts for ²²Na⁺ on the filter are related to the counts for ³H⁺ used as internal standard for the nonspecific binding. The ²²Na⁺/³H⁺ ratio (counts per filter) after filtration of 0.4 ml incubation media are shown for the samples of different compositions. A, in the absence of added oligomycin: (1) no protein, nonspecific binding without washing of the filter; (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.

Figure 5

Interactions of K⁺and BeF_xcomplex monitored by RH421 fluorescence.Inset (A) illustrates changes in RH421 fluorescence in response to addition of ligands to pig kidney enzyme expressed as percentage of the initial level of fluorescence. The K-induced responses were fit with a monoexponential function. Data from 3 to 4 individual experiments as well as the mean value ± SD are shown in A and B. A, the amplitude of the fluorescence change induced by addition of varying K⁺-concentration to preformed Na⁺,K⁺-ATPase–BeF_x complex. B, the observed rate constant of the fluorescence change (k_obs) as function on K⁺ concentration.

Figure 6

Structural rearrangements following Rb⁺binding to E2–BeF_x.A, open E2–BeF_x Mg²⁺-bound form (this study). Mg²⁺ is shown as a green sphere, and the positions of site I and II are indicated by gray dotted spheres. B, initial binding form (10 mM Rb⁺, 20 s). The 4-σ anomalous difference map is shown as a purple mesh, and cation-binding sites are shown as in A. Mg²⁺ site from open E2–BeF_x state is shown as green dotted sphere. C, early (Rb)E2–BeF_x form (soaked with 50 mM for 3 h, protomer 1). The 4-σ anomalous difference map is shown as a purple mesh, and cation-binding sites are shown as in A. D, late (Rb)E2–BeF_x form (soaked with 50 mM for 3 h, protomer 2). The anomalous difference map is contoured at 3σ (red mesh), and cation-binding sites are shown as in A. E, occluded [Rb₂]E2–MgF_x form (Protein Data Bank [PDB] ID: 3KDP) (6). Rb⁺ ions are shown as purple spheres.

Figure 7

Sequential structural changes leading to occlusion of Rb⁺in the E2 state. Structural differences (A and B) between the open E2–BeF_x Mg²⁺ (blue) and initial binding (Rb)E2–BeF_x form (green) in an overall view (A) and for the transmembrane domain (B), respectively, (C and D) the initial (Rb)E2–BeF_x form (green) and the early (Rb)E2–BeF_x (gold), (E and F) early (Rb)E2–BeF_x (gold) and late (Rb)E2–BeF_x form (orange), (G and H) late (Rb)E2–BeF_x form (orange) and fully occluded [Rb₂]E2–MgF_x (gray) (Protein Data Bank [PDB] ID: 3KDP (6)), and (I and J) open E2–BeF_x Mg²⁺ (blue) and fully occluded [Rb₂]E2–MgF_x (gray). All structures were aligned on αM7–M10. For clarity, only the regions showing major conformational rearrangements between the states have been highlighted.

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.

Figure 9

ADP from the [Na₃]E1–AlF₄⁻-ADP structure (Protein Data Bank [PDB] ID:3WGV (34)) modeled into the E2–BeF_xcomplex. N-domain alignment of the E2–BeF_x and the [Na₃]E1–AlF₄⁻-ADP structures showed a good fit for ADP at the nucleotide site with only few readjustment needed.