Long-Range Effects of Na+ Binding in Na,K-ATPase Reported by ATP

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 C-13 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 E-1 AlF4 ADP 3Na(+) form, the coupling between the ion binding sites and the nucleotide side is mediated by, among others, the M5 helix.