期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：6
页码：1755-1760
DOI：10.1073/pnas.1422997112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceNa+,K+-ATPase is the ion pump responsible for maintenance of the electrochemical gradients of Na+ and K+ across the membrane of animal cells. Cardiotonic steroids constitute a broad class of specific Na+,K+-ATPase inhibitors, including drugs of clinical importance with multiple physiological effects. The existence of several endogenous cardiotonic steroids suggests their involvement in health and disease mediated by various signaling pathways, but the structure-activity relationships are not yet understood. Using X-ray crystallography and analysis of binding kinetics, we characterize Na+,K+-ATPase complexes with a total of five cardiotonic steroids, showing variations in glycosylation, steroid core substituents, and structure of the lactone substituent. This insight is highly relevant for the understanding of physiological effects and future drug development based on cardiotonic steroids. 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, M4 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 M4 and hypothesize that winding/unwinding of M4 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 M1-2.
