Structures and characterization of digoxin- and bufalin-bound Na+,K+-ATPase compared with the ouabain-bound complex

Abstract

Cardiotonic steroids (CTSs) are specific and potent inhibitors of the Na(+),K(+)-ATPase, with highest affinity to the phosphoenzyme (E2P) forms. CTSs are comprised of a steroid core, which can be glycosylated, and a varying number of substituents, including a five- or six-membered lactone. These functionalities have specific influence on the binding properties. We report crystal structures of the Na(+),K(+)-ATPase in the E2P form in complex with bufalin (a nonglycosylated CTS with a six-membered lactone) and digoxin (a trisaccharide-conjugated CTS with a five-membered lactone) and compare their characteristics and binding kinetics with the previously described E2P-ouabain complex to derive specific details and the general mechanism of CTS binding and inhibition. CTSs block the extracellular cation exchange pathway, and cation-binding sites I and II are differently occupied: A single Mg(2+) is bound in site II of the digoxin and ouabain complexes, whereas both sites are occupied by K(+) in the E2P-bufalin complex. In all complexes, αM4 adopts a wound form, characteristic for the E2P state and favorable for high-affinity CTS binding. We conclude that the occupants of the cation-binding site and the type of the lactone substituent determine the arrangement of αM4 and hypothesize that winding/unwinding of αM4 represents a trigger for high-affinity CTS binding. We find that the level of glycosylation affects the depth of CTS binding and that the steroid core substituents fine tune the configuration of transmembrane helices αM1-2.

Keywords: Na/K-ATPase; cardiac glycosides; inhibitor; phosphoenzyme; structure.

Fig. 1.

Structural comparison of the crystal structures of the high-affinity Na⁺,K⁺-ATPase α₁β₁γ E2P–CTS complexes. The phosphoenzyme stabilized by bufalin, digoxin, and ouabain (5) is depicted in blue, green, and gray cartoons, respectively, and the bufalin, digoxin, and ouabain molecules are represented by magenta, orange, and dark gray sticks, respectively. The K⁺ and Mg²⁺ ions are represented by purple and yellow spheres, respectively. (A) Structural representation of the CTSs digoxin, bufalin, and ouabain. (B and C) The final 2F_o-F_c electron density maps of the E2P–bufalin and E2P–digoxin, respectively, complexes (contoured at 1.0σ level). The maps are represented by gray mesh. (D) Structural alignment of the E2P–bufalin, E2P–digoxin, and E2P–ouabain complexes performed on the segments αM7–10 showing a high degree of overall structural similarity. (E) The CTS-binding site visualized from the extracellular site based on the same alignment as above. The alignment reveals similar hydrophobic interactions between the α-surface of the CTS core and αM4–6. In contrast, different interactions are formed between the substituents at the β-surface of the CTS core and αM1–2, leading to minor CTS-induced rearrangements. (F) The CTS-binding site visualized from αM1–2. αM4 overlays well for Mg²⁺-bound complexes of E2P–digoxin and E2P–ouabain as well as the E2P–bufalin complex, despite potassium bound in the cation-binding sites.

Fig. 2.

The CTS- and cation-binding sites. The Na⁺,K⁺-ATPase α- and β-subunits are depicted in gray and black cartoons, respectively. The F_o-F_c map (contoured at 3.5σ level) obtained after initial rigid body refinement is depicted by beige mesh. (A) The digoxin-binding site. Digoxin and the side chains lining the binding cavity are depicted by orange and green sticks, respectively. The Mg²⁺ ion occupying cation site II is represented by a yellow sphere. (B) The bufalin-binding site. Bufalin and the side chains interacting with bufalin or the two bound potassium ions (purple spheres) are depicted by magenta and blue sticks, respectively. The anomalous difference Fourier map (contoured at 3.5σ level) obtained by replacing 100 mM KCl with 100 mM RbCl is depicted by orange mesh.

Fig. 3.

Interactions of the Na⁺,K⁺-ATPase with CTSs. (A) Binding of CTSs under steady-state conditions of the Na⁺,K⁺-ATPase reaction followed by a coupled enzymes method. Inset shows changes in the degree of ATPase inhibition over time after ouabain addition (second arrow). The rate constants k_obs were calculated as described in Materials and Methods and presented for ouabain (●) and bufalin (■) as functions of CTS concentrations. The data were fitted to a hyperbolic function, and parameters of the best approximation are presented in Table S2. (B) Effect of glycosylation on CTS binding to the Na⁺,K⁺-ATPase. The decrease of Na⁺,K⁺-ATPase activity is shown as a function of digoxin (△), ouabain (○), or ouabagenin (■) concentrations in the preincubation medium containing 3 mM P_i and 3 mM MgCl₂. The data points for digoxin and ouabain are almost overlapping. The data were fitted to a square root equation as described in ref. . The calculated K_d values are 2.8 ± 2 nM (digoxin), 1.1 ± 1 nM (ouabain), and 844 ± 100 nM (ouabagenin). (C) The effect of the size of lactone on binding of aglycones to the Na⁺,K⁺-ATPase. The decrease of Na⁺,K⁺-ATPase activity is shown as function of digitoxigenin (●) or bufalin (■) concentrations. Presence of 200 mM K⁺ affects the inhibitory properties of the aglycones (○ and □, respectively). The calculated K_d values are 14 ± 5 nM (bufalin), 9 ± 10 nM (bufalin; 200 mM K⁺), 26 ± 15 nM (digitoxigenin), and 650 ± 400 nM (digitoxigenin; 200 mM K⁺).