title: ATP and magnesium drive conformational changes of the Na+/K+-ATPase cytoplasmic headpiece.
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. Mg(2+) 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 Ca(2+)-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 Mg(2+) only, or in the simultaneous presence of Mg(2+) and ATP. Binding of the ATP alone (i.e. in the absence of Mg(2+)) 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 Mg(2+) 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 Mg(2+) binding to the enzyme-ATP complex induces in turn conformational change traditionally attributed to E1-->E2 transition.
authors.affiliation: Institute of Physiology, Academy of Sciences of the Czech Republic, Vídenská 1083, 14220 Prague 4, Czech Republic.
authors.forename: Lenka
authors.initials: L
authors.lastname: Grycova
authors.forename: Petr
authors.initials: P
authors.lastname: Sklenovsky
authors.forename: Zdenek
authors.initials: Z
authors.lastname: Lansky
authors.forename: Marika
authors.initials: M
authors.lastname: Janovska
authors.forename: Michal
authors.initials: M
authors.lastname: Otyepka
authors.forename: Evzen
authors.initials: E
authors.lastname: Amler
authors.forename: Jan
authors.initials: J
authors.lastname: Teisinger
authors.forename: Martin
authors.initials: M
authors.lastname: Kubala
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D055592:Biophysical Phenomena; D017931:DNA Primers / Q000235:genetics; D005454:Fluorescence Polarization; D066298:In Vitro Techniques; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology*; D051379:Mice; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
chemical_list: D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014364:Tryptophan; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium