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

Abstract

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 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 Mg2+, and crystallographic studies indicate that Rb +and Mn2+, but not Na+, bind to this site. Comparison with the low-affinity [K2]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 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+-Mg2+competition and explain the well-known antagonistic effect of K+on CTS binding.