Molecular Structure of the Na+,K+-ATPase α4β1 Isoform in Its Ouabain-Bound Conformation

Abstract

Na⁺,K⁺-ATPase is the active ion transport system that maintains the electrochemical gradients for Na⁺ and K⁺ across the plasma membrane of most animal cells. Na⁺,K⁺-ATPase is constituted by the association of two major subunits, a catalytic α and a glycosylated β subunit, both of which exist as different isoforms (in mammals known as α1, α2, α3, α4, β1, β2 and β3). Na⁺,K⁺-ATPase α and β isoforms assemble in different combinations to produce various isozymes with tissue specific expression and distinct biochemical properties. Na⁺,K⁺-ATPase α4β1 is only found in male germ cells of the testis and is mainly expressed in the sperm flagellum, where it plays a critical role in sperm motility and male fertility. Here, we report the molecular structure of Na⁺,K⁺-ATPase α4β1 at 2.37 Å resolution in the ouabain-bound state and in the presence of beryllium fluoride. Overall, Na⁺,K⁺-ATPase α4 structure exhibits the basic major domains of a P-Type ATPase, resembling Na⁺,K⁺-ATPase α1, but has differences specific to its distinct sequence. Dissimilarities include the site where the inhibitor ouabain binds. Molecular simulations indicate that glycosphingolipids can bind to a putative glycosphingolipid binding site, which could potentially modulate Na⁺,K⁺-ATPase α4 activity. This is the first experimental evidence for the structure of Na⁺,K⁺-ATPase α4β1. These data provide a template that will aid in better understanding the function Na⁺,K⁺-ATPase α4β1 and will be important for the design and development of compounds that can modulate Na⁺,K⁺-ATPase α4 activity for the purpose of improving male fertility or to achieve male contraception.

Keywords: Na+,K+-ATPase α4; cryoelectron microscopy; isoform; isozyme.

Figure 1

Structure of human Na⁺,K⁺-ATPase α4 with bound ouabain. (a) Overall cryo-EM structure of α4 in the ouabain-bound E2P state, viewed from the membrane plane showing the Na+,K+-ATPase α4 (blue) and β subunit (gray). Yellow density and stick models correspond to the detergent (GDN) and lipids (DOPC and cholesterol) present. N-linked glycans are shown as spheres. The membrane position (exoplasmic side-up) is indicated by a gray box. (b,c). Density map in the region surrounding the ouabain-binding sites (b) and in the P domain, where BeF3 binds to the conserved P-type ATPase aspartic acid (c). (d) Comparison of the α4 model (blue) with α1 in the same conformation (light gray, ouabain-bound E2P, 7wyt). (e) Clipped membrane slice of ouabain and GDN bound region at the exoplasmic surface of the membrane. Ouabain (green) and GDN (yellow) are shown as spheres. TM1 and TM2 are shown as transparent helixes for clarity. (f) Close-up view of the ouabain-binding site of Na⁺,K⁺-ATPase α4. Transparent sphere shows van der Waals volume of ouabain.

Figure 2

Ouabain-binding site. (a,b) Molecular details of ouabain-binding viewed from the direction in which TM1 and TM2 are located (a) and that of TM3 and TM4 (b), parallel to the membrane with exoplasmic side-up. TM1, TM2 or TM3 and TM4 are removed in each figure for clarity. Dotted lines indicate hydrogen bonds. Red spheres show water molecules. (c,d) Structure of Na⁺,K⁺-ATPase a1 (gray, 7wyt) superimposed to the structures in a and b. Only residues different from Na⁺,K⁺-ATPase a1 and Na⁺,K⁺-ATPase a4 are indicated in parentheses (numbering corresponding to Na⁺,K⁺-ATPase a1). (e) Schematic representations of ouabain binding. Residues that are located 3.9 Å from ouabain are shown. Expected hydrogen bonds within 3.3 Å are depicted as dotted lines.

Figure 3

GDN-binding site in Na⁺,K⁺-ATPase α4 (a,b). Surface representation of Na⁺,K⁺-ATPase α4 (a) and α1 ((b), pdb: 7wyt) viewed from parallel to the membrane with exoplasmic side-up. Sterol skeleton of GDN (orange sticks) is bound to the longitudinal crevice formed between TM2 and TM9-10 loop, and one of the maltose branches is incorporated to the side fenestration in α4 (a), while phosphatidylcholine (PC, gray sticks) is bound to the corresponding position in α1 (b). We also modeled another GDN molecule (only the hydrophobic part) and cholesterol (Chol) in the TM peripheral region (a). (c) Comparison of different rotamer conformation between Na⁺,K⁺-ATPase α1 (gray) and Na⁺,K⁺-ATPase α4 (blue). Amino acids from Na⁺,K⁺-ATPase α1 are indicated in parentheses. (d) Close-up view of maltose moieties. Dotted lines connect polar atoms located within 3.5 Å distance.

Figure 4

The binding of (a) GDN in the cryo-EM structure (b) GM1 in the final snapshot of one simulation replica and (c) GM3 in the final snapshot of one simulation replica. GM3 binds in a similar configuration in another replica. The binding mode of GM3 is almost identical to GDN. The graphs show the radial distribution functions (histograms of distances) between the lipid head group and the amino acids. The radial distribution functions show that GM3 binds close to residues K288, V982 and Y139. Note that the representation of the amino acids is coarse-grained in (b,c).