STRUCTURE-FUNCTION-RELATIONSHIPS OF E-1-E-2 TRANSITIONS AND CATION-BINDING IN NA,K-PUMP PROTEIN

Fully active Na,K-ATPase and lethal mutations can be expressed in yeas t cells in yields allowing far equilibrium ATP binding, occlusion of T 1(+), K+ displacement of ATP, and Na+-dependent phosphorylation with d eterminations of affinity constants for binding and constants for the conformational equilibria. Removal of the charge and hydrophobic subst itution of the phosphorylated residue (Asp(369)Ala) reveals an intrins ic high affinity for ATP binding (K-d 2.8 vs. 100 nM for wild type) an d causes a shift of conformational equilibrium towards the E-2 form. S ubstitution of Glu(327), Glu(779), Asp(804) or Asp(808) in transmembra ne segments 4, 5, and 6 shows that each of these residues are essentia l for high-affinity occlusion of K+ and for binding of Na+. Substituti on of other residues in segment 5 shows that the carboxamide group of Asn(776) is important for binding of both K+ and Na+. Differential eff ects of the relevant mutations identify Thr(774) as specific determina nt 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 t he helix during the E-1-E-2 transition. (C) 1998 Elsevier Science B.V.