Abstract
Conformational changes of the Na+/K+-ATPase isolated large cytoplasmic segment connecting transmembrane helices M4 and M5 (C45) induced by the interaction with enzyme ligands (i.e. Mg2+ and/or ATP) were investigated by means of the intrinsic tryptophan fluorescence measurement and molecular dynamic simulations.
Our data revealed that this model system consisting of only two domains retained the ability to adopt open or closed conformation, i.e. behavior, which is expected from the crystal structures of relative Ca2+-ATPase from sarco(endo)plasmic reticulum for the corresponding part of the entire enzyme. Our data revealed that the C45 is found in the closed conformation in the absence of any ligand, in the presence of Mg2+ only, or in the simultaneous presence of Mg2+ and ATP.
Binding of the ATP alone (i.e. in the absence of Mg2+) induced open conformation of the C45. The fact that the transmembrane part of the enzyme was absent in our experiments suggested that the observed conformational changes are consequences only of the interaction with ATP or Mg2+ and may not be related to the transported cations binding/ release, as generally believed.
Our data are consistent with the model, where ATP binding to the low-affinity site induces conformational change of the cytoplasmic part of the enzyme, traditionally attributed to E2 -> E1 transition, and subsequent Mg2+ binding to the enzyme-ATP complex induces in turn conformational change traditionally attributed to E1 -> E2 transition.