title: 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 Mg(2+), and crystallographic studies indicate that Rb(+) and Mn(2+), but not Na(+), bind to this site. Comparison with the low-affinity [K2]E2-MgF(x)-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 Mg(2+). 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(+)-Mg(2+) competition and explain the well-known antagonistic effect of K(+) on CTS binding.
authors.affiliation: Centre for Membrane Pumps in Cells and Disease (PUMPkin), Danish National Research Foundation, DK-8000 Aarhus C, Denmark.
authors.forename: Mette
authors.initials: M
authors.lastname: Laursen
authors.forename: Laure
authors.initials: L
authors.lastname: Yatime
authors.forename: Poul
authors.initials: P
authors.lastname: Nissen
authors.forename: Natalya U
authors.initials: NU
authors.lastname: Fedosova
mesh_terms: D000818:Animals; D001665:Binding Sites; D002316:Cardiotonic Agents / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D010042:Ouabain / Q000737:chemistry* / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism; D013552:Swine
chemical_list: D002316:Cardiotonic Agents; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D011188:Potassium
keywords: cardiac glycosides; crystallography; membrane proteins; phosphoenzyme