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

TY - JOUR

T1 - Structure-function relationships of E1-E2 transitions and cation binding in Na,K-pump protein

AU - Jorgensen, Peter L.

AU - Nielsen, Jesper M.

AU - Rasmussen, Jakob H.

AU - Pedersen, Per Amstrup

PY - 1998/6/10

Y1 - 1998/6/10

N2 - Fully active Na,K-ATPase and lethal mutations can be expressed in yeast cells in yields allowing for 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 (Asp369Ala) 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 E2 form. Substitution of Glu327, Glu779 Asp804 or Asp808 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 Asn776 is important for binding of both K+ and Na+. Differential effects of the relevant mutations identify Thr774 as specific determinant of Na+ binding in the E1P[3Na] form, whereas Ser775 is a specific participant of high-affinity binding of the E2[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 E1-E2 transition.

AB - Fully active Na,K-ATPase and lethal mutations can be expressed in yeast cells in yields allowing for 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 (Asp369Ala) 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 E2 form. Substitution of Glu327, Glu779 Asp804 or Asp808 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 Asn776 is important for binding of both K+ and Na+. Differential effects of the relevant mutations identify Thr774 as specific determinant of Na+ binding in the E1P[3Na] form, whereas Ser775 is a specific participant of high-affinity binding of the E2[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 E1-E2 transition.

KW - (Kidney)

KW - ATP binding

KW - Conformational transition

KW - Heterologous expression

KW - K binding

KW - K occlusion

KW - Mutation

KW - Na,K-ATPase

KW - Na binding

KW - Na-dependent phosphorylation

KW - Yeast cell

UR - http://www.scopus.com/inward/record.url?scp=0032311442&partnerID=8YFLogxK

U2 - 10.1016/S0005-2728(98)00043-7

DO - 10.1016/S0005-2728(98)00043-7

M3 - Journal article

C2 - 9693723

AN - SCOPUS:0032311442

SN - 0005-2728

VL - 1365

SP - 65

EP - 70

JO - B B A - Bioenergetics

JF - B B A - Bioenergetics

IS - 1-2

ER -