Abstract
A cytoplasmic nontransport K(+)/Rb(+) site in the P-domain of the Na(+), K(+)-ATPase has been identified by anomalous difference Fourier map analysis of crystals of the [Rb(2)].E(2).MgF(4)(2-) form of the enzyme. The functional roles of this third K(+)/Rb(+) binding site were studied by site-directed mutagenesis, replacing the side chain of Asp(742) donating oxygen ligand(s) to the site with alanine, glutamate, and lysine. Unlike the wild-type Na(+), K(+)-ATPase, the mutants display a biphasic K(+) concentration dependence of E(2)P dephosphorylation, indicating that the cytoplasmic K(+) site is involved in activation of dephosphorylation. The affinity of the site is lowered significantly (30-200-fold) by the mutations, the lysine mutation being most disruptive. Moreover, the mutations accelerate the E(2) to E(1) conformational transition, again with the lysine substitution resulting in the largest effect. Hence, occupation of the cytoplasmic K(+)/Rb(+) site not only enhances E(2)P dephosphorylation but also stabilizes the E(2) dephosphoenzyme. These characteristics of the previously unrecognized nontransport site make it possible to account for the hitherto poorly understood trans-effects of cytoplasmic K(+) by the consecutive transport model, without implicating a simultaneous exposure of the transport sites toward the cytoplasmic and extracellular sides of the membrane. The cytoplasmic K(+)/Rb(+) site appears to be conserved among Na(+), K(+)-ATPases and P-type ATPases in general, and its mode of operation may be associated with stabilizing the loop structure at the C-terminal end of the P6 helix of the P-domain, thereby affecting the function of highly conserved catalytic residues and promoting helix-helix interactions between the P- and A-domains in the E(2) state.
Highlights
The family of P-type ATPases characterized by the formation of a phosphorylated enzyme intermediate through transfer of the ␥-phosphate of ATP to a conserved aspartate residue in the enzyme
In sarco(endo)plasmic reticulum Ca2ϩ-ATPase (SERCA), the activity is under the influence of a cytoplasmic Kϩ site in the P-domain (20 –22), and a similar site has been identified in the plant plasma membrane Hϩ-ATPase by mutational analysis [23]
The Naϩ,Kϩ-ATPase was crystallized, and its structure was determined at 3.5 Å resolution in the [Rb2]1⁄7E21⁄7MgF42Ϫ form, where two Rbϩ ions are bound at the membranous transport sites in an occluded state [4]
Summary
M1–M10, transmembrane segments numbered from the N terminus; K0.5, ligand concentration giving half-maximum effect; SERCA, sarco(endo)plasmic reticulum Ca2ϩ-ATPase. It is of particular importance that cytoplasmic Kϩ has been shown to stimulate Naϩ transport toward the extracellular side (16 –18) as well as the processing of the phosphoenzyme involved in extracellular Naϩ release and dephosphorylation activated by extracellular Kϩ [18] To account for these effects, it seems necessary to postulate the presence of at least one cytoplasmically facing Kϩ site existing simultaneously with the two extracellularly facing Kϩ transport sites. We reveal the existence of such an additional (third) Rbϩ/Kϩ binding site in the structure of the Naϩ,Kϩ-ATPase ␣-subunit This site is located in the cytoplasmic part of the ␣-subunit, in the P-domain, and is, not directly involved in the binding of the translocated ions. The affinity of the site for cytoplasmic Kϩ is relatively high, and it is likely to be permanently saturated at the high internal Kϩ concentration present under physiological conditions
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