Elucidation of the Na+, K+-ATPase digitalis binding site

Here, we propose a binding mode for digoxin and several analogues to the Na+, K+-ATPase. A 3D-structural model of the extracellular loop regions of the catalytic α1-subunit of the digitalis-sensitive sheep Na+, K+-ATPase was constructed from the crystal structure of an E1Ca2+ conformation of the SERCA1a and a consensus orientation for digitalis binding was inferred from the in silico docking of a series of steroid-based cardiotonic compounds. Analyses of species-specific enzyme affinities for ouabain were also used to validate the model and, for the first time, propose a detailed model of the digitalis binding site.