Structure-function relationships of E1-E2 transitions and cation binding in Na,K-pump protein
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Structure-function relationships of E1-E2 transitions and cation binding in Na,K-pump protein. / Jorgensen, Peter L.; Nielsen, Jesper M.; Rasmussen, Jakob H.; Pedersen, Per Amstrup.
I: Biochimica et Biophysica Acta - Bioenergetics, Bind 1365, Nr. 1-2, 10.06.1998, s. 65-70.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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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
VL - 1365
SP - 65
EP - 70
JO - B B A - Bioenergetics
JF - B B A - Bioenergetics
SN - 0005-2728
IS - 1-2
ER -
ID: 227301737