Long-range effects of Na+ binding in Na,K-ATPase reported by ATP

Middleton, David Andrew and Fedosova, Natalya U. and Esmann, Mikael (2015) Long-range effects of Na+ binding in Na,K-ATPase reported by ATP. Biochemistry, 54 (47). pp. 7041-7047. ISSN 0006-2960

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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 13C cross-polarization magic-angle spinning (CP-MAS) solid-state NMR. 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+ are used to induce binding. Differences in chemical shifts for ATP carbon 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 for 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 analog eosin show that the dissociation rate constant for eosin is larger in Tris+ than in Na+, giving kinetic evidence for 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.

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This document is the Accepted Manuscript version of a Published Work that appeared in final form in Biochemistry, copyright ©2015 American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://pubs.acs.org/doi/10.1021/acs.biochem.5b00893
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11 Nov 2015 13:32
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31 Dec 2023 00:36