Reconstruction of the Complete Ouabain-binding Pocket of Na,K-ATPase in Gastric H,K-ATPase by Substitution of Only Seven Amino Acids

Abstract

Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na,KATPase), 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 (

The non-gastric H,K-ATPase as a tool to study the ouabain-binding site in Na,K-ATPase

“…The resulting model showed important roles for several of the amino acids that were identified in both studies with the chimeras [13,14]. In addition, the two amino acids discovered by Lingrel's group [15] were very important in the model.…”

“…The chimeras and mutants used in this study were: the rat Na,K-ATPase α 1 -subunit (wild type), the R111Q/ D122N mutant (QN) of this enzyme [10,11], the rat non-gastric H, K-ATPase α 2 -subunit [13] as well as its EGPLC mutant (D312E, S319G, A778P, I795L, F802C) [14]. Generation of the vectors containing the α 2 -subunit of rat non-gastric H,K-ATPase with the β 1 -subunit of rat Na,K-ATPase or the α 1 -subunit of rat Na,K-ATPase with the β 1 -subunit of sheep Na,K-ATPase that were suited for the baculovirus expression system, has been reported before [5,14].…”

“…The homology model of the QN mutant of rat Na,K-ATPase [13], based on an E 2 -P-like structure of Ca 2+ -ATPase (1WPG; [20]), was used for in silico docking with the FlexX program [21], version 1.13.5 L. FlexX predicts the conformations of a set of energetically favourable molecular complexes consisting of the ligand bound to the active site of the protein. The complexes were labelled with a score that approximates binding energy.…”

“…Similarly, as described earlier for ouabain [13], docking experiments were carried out with the five analogues in a homology model of the E 2 -P form of human Na,K-ATPase, based on the 1WPG structure of Ca 2+ -ATPase. The human ATPase is comparable to the QN mutant.…”

“…The four transmembrane segments originating from Na,K-ATPase still contained 48 amino acids that were specific for this enzyme. Later, we could reduce this number to seven and still kept a high affinity for ouabain [12,13]. In the latter studies, we used the oocyte expression system and restricted ourselves to the measurement of ouabain binding.…”