Structure-function relationships of Na+, K+, ATP, or Mg2+ binding and energy transduction in Na,K-ATPase

The focus of this article is on progress in establishing structure-function relationships through site-directed mutagenesis and direct binding assay o f Tl+, Rb+, K+, Na+, Mg2+ or free ATP at equilibrium in Na,K-ATPase. Direct binding may identify residues coordinating cations in the E-2[2K] or E1P[3 Na] forms of the ping-pong reaction sequence and allow estimates of their c ontributions to the change of Gibbs free energy of binding. This is require d to understand the molecular basis for the pronounced Na/K selectivity at the cytoplasmic and extracellular surfaces. Intramembrane Glu(327) in trans membrane segment M4, Glu(779) in M5, Asp(804) and Asp(808) in M6 are essent ial for tight binding of K+ and Na+. Asn(324) and Glu(327) in M4, Thr(774), Asn(776), and Glu(779) in 771-YTLTSNIPEITP of M5 contribute to Na+/K+ sele ctivity. Free ATP binding identifies Arg(544) as essential for high affinit y binding of ATP or ADP. In the 708-TGDGVND segment, mutations of Asp(710) or Asn(713) do not interfere with free ATP binding. Asp(710) is essential a nd Asn(713) is important for coordination of Mg2+ in the E1P[3Na] complex, but they do not contribute to Mg2+ binding in the E2P-ouabain complex. Tran sition to the E2P form involves a shift of Me2+ coordination away from Asp( 710) and Asn(713) and the two residues become more important for hydrolysis of the acyl phosphate bond at Asp(369). (C) 2001 Elsevier Science B.V. All rights reserved.