Structure-function relationships of E1-E2 transitions and cation binding in Na,K-pump protein

Fully active Na,K-ATPase and lethal mutations can be expressed in yeast cells in yields allowing far equilibrium ATP binding, occlusion of T1(+), K+ displacement of ATP, and Na+-dependent phosphorylation with determinations of affinity constants for binding and constants for the conformational equilibria. Removal of the charge and hydrophobic substitution of the phosphorylated residue (Asp(369)Ala) reveals an intrinsic high affinity for ATP binding (K-d 2.8 vs. 100 nM for wild type) and causes a shift of conformational equilibrium towards the E-2 form. Substitution of Glu(327), Glu(779), Asp(804) or Asp(808) in transmembrane segments 4, 5, and 6 shows that each of these residues are essential for high-affinity occlusion of K+ and for binding of Na+. Substitution of other residues in segment 5 shows that the carboxamide group of Asn(776) is important for binding of both K+ and Na+. Differential effects of the relevant mutations identify Thr(774) as specific determinant of Naf binding in the E1P[3Na] form, whereas Ser(775) is a specific participant of high-affinity binding of the E-2 [2K] form, suggesting that these residues engage in formation of a molecular Na+/K+ switch. The position of the switch may be controlled by rotating or tilting the helix during the E-1-E-2 transition. (C) 1998 Elsevier Science B.V.