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

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.