Crystal structure of the high-affinity Na ⁺ ,K ⁺ -ATPase–ouabain complex with Mg ²⁺ bound in the cation binding site

<jats:p> The Na <jats:sup>+</jats:sup> ,K <jats:sup>+</jats:sup> -ATPase maintains electrochemical gradients for Na <jats:sup>+</jats:sup> and K <jats:sup>+</jats:sup> 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 <jats:sup>+</jats:sup> ,K <jats:sup>+</jats:sup> -ATPase. Here we describe a crystal structure of the phosphorylated pig kidney Na <jats:sup>+</jats:sup> ,K <jats:sup>+</jats:sup> -ATPase in complex with the CTS representative ouabain, extending to 3.4 Å resolution. The structure provides key details on CTS binding, revealing an extensive hydrogen bonding network formed by the β-surface of the steroid core of ouabain and the side chains of αM1, αM2, and αM6. Furthermore, the structure reveals that cation transport site II is occupied by Mg <jats:sup>2+</jats:sup> , and crystallographic studies indicate that Rb <jats:sup>+</jats:sup> and Mn <jats:sup>2+</jats:sup> , but not Na <jats:sup>+</jats:sup> , bind to this site. Comparison with the low-affinity [K <jats:sub>2</jats:sub> ]E2–MgF <jats:sub>x</jats:sub> –ouabain structure [Ogawa et al. (2009) <jats:italic>Proc Natl Acad Sci USA</jats:italic> 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 Mg <jats:sup>2+</jats:sup> . K <jats:sup>+</jats:sup> binding at the same site unwinds a turn of αM4, 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 <jats:sup>+</jats:sup> –Mg <jats:sup>2+</jats:sup> competition and explain the well-known antagonistic effect of K <jats:sup>+</jats:sup> on CTS binding. </jats:p>