title: Structures and characterization of digoxin- and bufalin-bound Na+,K+-ATPase compared with the ouabain-bound complex.
abstract: Cardiotonic steroids (CTSs) are specific and potent inhibitors of the Na(+),K(+)-ATPase, with highest affinity to the phosphoenzyme (E2P) forms. CTSs are comprised of a steroid core, which can be glycosylated, and a varying number of substituents, including a five- or six-membered lactone. These functionalities have specific influence on the binding properties. We report crystal structures of the Na(+),K(+)-ATPase in the E2P form in complex with bufalin (a nonglycosylated CTS with a six-membered lactone) and digoxin (a trisaccharide-conjugated CTS with a five-membered lactone) and compare their characteristics and binding kinetics with the previously described E2P-ouabain complex to derive specific details and the general mechanism of CTS binding and inhibition. CTSs block the extracellular cation exchange pathway, and cation-binding sites I and II are differently occupied: A single Mg(2+) is bound in site II of the digoxin and ouabain complexes, whereas both sites are occupied by K(+) in the E2P-bufalin complex. In all complexes, αM4 adopts a wound form, characteristic for the E2P state and favorable for high-affinity CTS binding. We conclude that the occupants of the cation-binding site and the type of the lactone substituent determine the arrangement of αM4 and hypothesize that winding/unwinding of αM4 represents a trigger for high-affinity CTS binding. We find that the level of glycosylation affects the depth of CTS binding and that the steroid core substituents fine tune the configuration of transmembrane helices αM1-2.
authors.affiliation: Centre for Membrane Pumps in Cells and Disease, Danish National Research Foundation, DK-8000 Aarhus C, Denmark; Departments of Biomedicine and.
authors.forename: Mette
authors.initials: M
authors.lastname: Laursen
authors.affiliation: Centre for Membrane Pumps in Cells and Disease, Danish National Research Foundation, DK-8000 Aarhus C, Denmark; Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark; and.
authors.forename: Jonas Lindholt
authors.initials: JL
authors.lastname: Gregersen
authors.affiliation: Centre for Membrane Pumps in Cells and Disease, Danish National Research Foundation, DK-8000 Aarhus C, Denmark; Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark; and.
authors.forename: Laure
authors.initials: L
authors.lastname: Yatime
authors.affiliation: Centre for Membrane Pumps in Cells and Disease, Danish National Research Foundation, DK-8000 Aarhus C, Denmark; Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark; and Danish Research Institute for Translational Neuroscience, Nordic-European Molecular Biology Laboratory Partnership of Molecular Medicine, DK-8000 Aarhus C, Denmark pn@mbg.au.dk nf@biomed.au.dk.
authors.forename: Poul
authors.initials: P
authors.lastname: Nissen
authors.affiliation: Centre for Membrane Pumps in Cells and Disease, Danish National Research Foundation, DK-8000 Aarhus C, Denmark; Departments of Biomedicine and pn@mbg.au.dk nf@biomed.au.dk.
authors.forename: Natalya U
authors.initials: NU
authors.lastname: Fedosova
mesh_terms: D000818:Animals; D002018:Bufanolides / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004077:Digoxin / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D006031:Glycosylation; D007700:Kinetics; D008958:Models, Molecular; D010042:Ouabain / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013552:Swine; D014961:X-Ray Diffraction
chemical_list: D002018:Bufanolides; D010042:Ouabain; D004077:Digoxin; D000254:Sodium-Potassium-Exchanging ATPase; C022777:bufalin
keywords: Na/K-ATPase; cardiac glycosides; inhibitor; phosphoenzyme; structure