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Poul Nissen

Structures and characterization of digoxin- and bufalin-bound Na+,K+-ATPase compared with the ouabain-bound complex

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  • Mette Laursen, Centre for Membrane Pumps in Cells and Disease (PUMPKIN), Danish National Research Foundation, Denmark
  • Jonas Lindholt Gregersen, Denmark
  • Laure Yatime, Centre for Membrane Pumps in Cells and Disease (PUMPKIN), Danish National Research Foundation, Denmark
  • Poul Nissen
  • Natalya Fedosova
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 Mg2+ 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 substituent fine tune the configurationof transmembrane helices αM1–2.
Original languageEnglish
JournalPNAS (Proceedings of the National Academy of Sciences of the United States of America)
Pages (from-to)1755-1760
Number of pages6
Publication statusPublished - 10 Feb 2015

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