Reconstruction of the complete ouabain-binding pocket of Na,K-ATPase in gastric H,K-ATPase by substitution of only seven amino acids

Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na, K-ATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we started from the fact that a closely related enzyme, the gastric H, K-ATPase, does not bind glycosides like ouabain. Previously, we showed that a chimera of these two enzymes, in which only the M3-M4 and M5-M6 hairpins were of Na, K- ATPase, bound ouabain with high affinity (Koenderink, J. B., Hermsen, H. P. H., Swarts, H. G. P., Willems, P. H. G. M., and De Pont, J. J. H. H. M. ( 2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11209 11214). We also demonstrated that only three amino acids (Phe(783), Thr(797), and Asp(804)) present in the M5-M6 hairpin of Na,K-ATPase were sufficient to confer high affinity ouabain binding to a chimera which contained in addition the M3-M4 hairpin of Na, K-ATPase (Qiu, L. Y., Koenderink, J. B., Swarts, H. G., Willems, P. H., and De Pont, J. J. H. H. M. ( 2003) J. Biol. Chem. 278, 47240 - 47244). To further pinpoint the ouabain-binding site here we used a chimerabased loss-of-function strategy and identified four amino acids (Glu(312), Val(314), Ile(315), Gly(319)), all present in M4, as being important for ouabain binding. In a final gain-of-function study we showed that a gastric H, K- ATPase that contained Glu312, Val314, Ile315, Gly319, Phe783, Thr797, and Asp804 of Na, K- ATPase bound ouabain with the same affinity as the native enzyme. Based on the E2P crystal structure of Ca2+ -ATPase we constructed a homology model for the ouabain-binding site of Na, K- ATPase involving all seven amino acids as well as several earlier postulated amino acids.