Structural insights into the binding of cardiac glycosides to the digitalis receptor revealed by solid-state NMR

Several biologically active derivatives of the cardiotonic steroid ouabain have been made containing NMR isotopes (C-13, H-2, and F-19) in the rhamnos e sugar and steroid moieties, and examined at the digitalis receptor site o f renal Na+/K+-ATPase by a combination of solid-state NMR methods. Deuteriu m NMR spectra of H-2-labeled inhibitors revealed that the sugar group was o nly loosely associated with the binding site, whereas the steroid group was more constrained, probably because of hydrogen bonding to residues around the K+-channel region. Crosspolarization magic-angle spinning NMR showed th at chemical shifts of inhibitors C-13-tabeled in the sugar group moved down field by 0.5 ppm after binding to the digitalis site, suggesting that the s ugar was close to aromatic side groups. A(19)F, C-13- rotational-echo doubl e-resonance NMR strategy was used to determine the structure of an inhibito r in the digitalis receptor site, and it showed that the ouabain derivative s adopt a conformation in which the sugar extends out of the plane of the s teroid ring system. The combined structural and dynamic information favors a model for inhibition in which the ouabain analogues lie across the surfac e of the Na+/K+-ATPase alpha -subunit with the sugar group facing away from the surface of the membrane but free to move into contact with one or more aromatic residues.