Crystal structure of the high-affinity Na+,K+-ATPase-ouabain complex with Mg2+ bound in the cation binding site

The Na+,K+-ATPase maintains electrochemical gradients for Na+ and K+ that are critical for animal cells. Cardiotonic steroids (CTSs), widely used in the clinic and recently assigned a role as endogenous regulators of intracellular processes, are highly specific inhibitors of the Na+,K+-ATPase. Here we describe a crystal structure of the phosphorylated pig kidney Na+,K+-ATPase in complex with the CTS representative ouabain, extending to 3.4 angstrom resolution. The structure provides key details on CTS binding, revealing an extensive hydrogen bonding network formed by the beta-surface of the steroid core of ouabain and the side chains of alpha M1, alpha M2, and alpha M6. Furthermore, the structure reveals that cation transport site II is occupied by Mg2+, and crystallographic studies indicate that Rb+ and Mn2+, but not Na+, bind to this site. Comparison with the low-affinity [K-2]E2-MgFx-ouabain structure [Ogawa et al. (2009) Proc Natl Acad Sci USA 106(33):13742-13747) shows that the CTS binding pocket of [Mg]E2P allows deep ouabain binding with possible long-range interactions between its polarized five-membered lactone ring and the Mg2+. K+ binding at the same site unwinds a turn of alpha M4, dragging residues Ile318-Val325 toward the cation site and thereby hindering deep ouabain binding. Thus, the structural data establish a basis for the interpretation of the biochemical evidence pointing at direct K+-Mg2+ competition and explain the well-known antagonistic effect of K+ on CTS binding.