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

David Middleton, David A. Middleton, Saffron Rankin, Mikael Esmann, Anthony Watts

    Research output: Contribution to journalArticlepeer-review

    Abstract

    Several biologically active derivatives of the cardiotonic steroid ouabain have been made containing NMR isotopes (13C, 2H, and 19F) in the rhamnose sugar and steroid moieties, and examined at the digitalis receptor site of renal Na+/K+-ATPase by a combination of solid-state NMR methods. Deuterium NMR spectra of 2H-labeled inhibitors revealed that the sugar group was only 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 that chemical shifts of inhibitors 13C-labeled in the sugar group moved downfield by 0.5 ppm after binding to the digitalis site, suggesting that the sugar was close to aromatic side groups. A 19F, 13C- rotational-echo double-resonance NMR strategy was used to determine the structure of an inhibitor in the digitalis receptor site, and it showed that the ouabain derivatives adopt a conformation in which the sugar extends out of the plane of the steroid ring system. The combined structural and dynamic informarion favors a model for inhibition in which the ouabain analogues lie across the surface of the Na+/K+-ATPase α-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.
    Original languageEnglish
    Pages (from-to)13602-13607
    Number of pages5
    JournalProceedings of the National Academy of Sciences of the United States of America
    Volume97
    Issue number25
    Publication statusPublished - 5 Dec 2000

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