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 of 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[3Na] forms of the ping-pong reaction sequence and allow estimates of their contributions to the change of Gibbs free energy of binding. This is required to understand the molecular basis for the pronounced Na/K selectivity at the cytoplasmic and extracellular surfaces. Intramembrane Glu(327) in transmembrane segment M4, Glu(779) in M5, Asp(804) and Asp(808) in M6 are essential 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+ selectivity. Free ATP binding identifies Arg(544) as essential for high affinity 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 and 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. Transition 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.