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

Abstract 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 + , Mg 2+ or free ATP at equilibrium in Na,K-ATPase. Direct binding may identify residues coordinating cations in the E 2 [2K] or E 1 P[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 Mg 2+ in the E 1 P[3Na] complex, but they do not contribute to Mg 2+ binding in the E 2 P-ouabain complex. Transition to the E 2 P form involves a shift of Mg 2+ 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 .