Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain

Abstract

The sodium-potassium pump (Na(+),K(+)-ATPase) is responsible for establishing Na(+) and K(+) concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na(+),K(+)-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 A resolution in a state analogous to E2.2K(+).Pi. Ouabain is deeply inserted into the transmembrane domain with the lactone ring very close to the bound K(+), in marked contrast to previous models. Due to antagonism between ouabain and K(+), the structure represents a low-affinity ouabain-bound state. Yet, most of the mutagenesis data obtained with the high-affinity state are readily explained by the present crystal structure, indicating that the binding site for ouabain is essentially the same. According to a homology model for the high affinity state, it is a closure of the binding cavity that confers a high affinity.

Fig. 1.

Crystal structure of Na⁺,K⁺-ATPase with bound ouabain. (A) Superimposition of Cα traces of Na⁺,K⁺-ATPase from shark rectal gland in ouabain-bound (yellow) and ouabain-unbound (cyan) forms, viewed along the membrane plane. Na⁺,K⁺-ATPase is fixed in a state analogous to E2·2K⁺·Pi, with MgF₄²⁻ as a stable phosphate analog. Ouabain (OBN; green and red) and K⁺ ions (I, II, and c; purple) are shown in space fill. The β-subunit, FXYD protein, 3 cytoplasmic domains (A, N, and P), and several transmembrane helices are marked. Green horizontal lines indicate the approximate position of lipid bilayer (M). (B) An F_obs - F_calc omit-annealed map of ouabain at 3σ, superimposed on the final atomic model. The underscores indicate that the residues have been identified as affecting ouabain binding by mutagenesis. (C) A diagram of ouabain.

Fig. 2.

Structural changes in the transmembrane region of Na⁺,K⁺-ATPase due to ouabain binding and corresponding changes in Ca²⁺-ATPase. (A) Superimposition of Cα traces of Na⁺,K⁺-ATPase in ouabain-bound (yellow) and ouabain-unbound (cyan) forms, viewed parallel to the membrane plane in approximately orthogonal direction to Fig. 1A. Ouabain (OBN; green and red) is shown in stick, and K⁺ ions (I and II; purple) are shown in space fill. Locations of Gly residues involved in structural changes are marked with small balls. M1 and M2 helices are cut away for clarity. (B and C) Superimposition of transmembrane helices (cylinders) viewed approximately perpendicular to the membrane plane from the extracellular side. (B) Na⁺,K⁺-ATPase in the state analogous to E2·2K⁺·Pi (cyan) and that with bound ouabain at low affinity (yellow) and a hypothetical one for high-affinity ouabain-bound state (E2P, orange). (C) Ca²⁺-ATPase in the states analogous to the E2·Pi product state (green; PDB ID, 1WPG) and the E2P ground state (orange; PDB ID, 2ZBE). Arrows show the movements of transmembrane helices accompanying the E2·Pi → E2P transition.

Fig. 3.

Binding of ouabain to the transmembrane binding site, viewed approximately perpendicular to the membrane plane from the extracellular side. Water accessible surface superimposed on the atomic model. (A) Ouabain-unbound state. (B) Low-affinity ouabain-bound state. (C) Hypothetical one for high-affinity ouabain-bound state. Dotted orange lines show likely hydrogen bonds. Dotted yellow line in panel A shows the outline of ouabain placed at the same position in panel B. The α- and β-sides and van der Waals surface of ouabain (green and red) are shown in panels B and C.

Fig. 4.

Structural changes around the M4–M6 transmembrane helices on ouabain binding. Viewed in stereo approximately parallel to the membrane. Water accessible surface in atom color (A, ouabain-bound with low affinity; B, ouabain-unbound) superimposed with the respective atomic models. Ouabain (OBN; green and red sticks) in panel B is placed at the same position as in panel A. Green dotted lines show likely hydrogen bonds.

Fig. 5.

Structural changes between the E2P ground state and the E2·Pi product state observed in the crystal structures of Ca²⁺-ATPase. Viewed approximately perpendicular to the membrane plane from the extracellular side. Water accessible surface superimposed on the atomic model. (A) E2·Pi product state [E2·MgF₄²⁻(TG); PDB ID: 1WPG]. (B) E2P ground state (E2·BeF₃⁻; PDB ID: 2ZBE). Atomic models of ouabain are placed in the positions corresponding to that observed in the Na⁺,K⁺-ATPase crystal structure (Fig. 3B).

Fig. 6.

Details of the ouabain binding site. (A) Crystal structure of the low-affinity ouabain (OBN)-bound state. (B) Hypothetical one for the high-affinity ouabain-bound state. Viewed in stereo approximately parallel to the membrane. The B and D rings and the α- and the β-sides of ouabain are identified. Orange dotted lines show likely hydrogen bonds.