title: Long-Range Effects of Na(+) Binding in Na,K-ATPase Reported by ATP.
abstract: This paper addresses the question of long-range interactions between the intramembranous cation binding sites and the cytoplasmic nucleotide binding site of the ubiquitous ion-transporting Na,K-ATPase using (13)C cross-polarization magic-angle spinning (CP-MAS) solid-state nuclear magnetic resonance. High-affinity ATP binding is induced by the presence of Na(+) as well as of Na-like substances such as Tris(+), and these ions are equally efficient promoters of nucleotide binding. CP-MAS analysis of bound ATP with Na,K-ATPase purified from pig kidney membranes reveals subtle differences in the nucleotide interactions within the nucleotide site depending on whether Na(+) or Tris(+) is used to induce binding. Differences in chemical shifts for ATP atoms C1' and C5' observed in the presence of Na(+) or Tris(+) suggest alterations in the residues surrounding the bound nucleotide, hydrogen bonding, and/or conformation of the ribose ring. This is taken as evidence of a long-distance communication between the Na(+)-filled ion sites in the membrane interior and the nucleotide binding site in the cytoplasmic domain and reflects the first conformational change ultimately leading to phosphorylation of the enzyme. Stopped-flow fluorescence measurements with the nucleotide analogue eosin show that the dissociation rate constant for eosin is larger in Tris(+) than in Na(+), giving kinetic evidence of the difference in structural effects of Na(+) and Tris(+). According to the recent crystal structure of the E1·AlF4(-)·ADP·3Na(+) form, the coupling between the ion binding sites and the nucleotide side is mediated by, among others, the M5 helix.
authors.affiliation: Department of Chemistry, B8 Faraday Building, Lancaster University , Lancaster LA1 4YB, U.K.
authors.forename: David A
authors.initials: DA
authors.lastname: Middleton
authors.affiliation: Department of Biomedicine, University of Aarhus , Ole Worms Allé 6, DK-8000 Aarhus C, Denmark.
authors.forename: Natalya U
authors.initials: NU
authors.lastname: Fedosova
authors.affiliation: Department of Biomedicine, University of Aarhus , Ole Worms Allé 6, DK-8000 Aarhus C, Denmark.
authors.forename: Mikael
authors.initials: M
authors.lastname: Esmann
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002414:Cations, Monovalent / Q000378:metabolism; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011188:Potassium / Q000378:metabolism; D011485:Protein Binding; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D013552:Swine
chemical_list: D002414:Cations, Monovalent; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium