Abstract
The consequences of mutations Ile(265) --> Ala, Thr(267) --> Ala, Gly(271) --> Ala, and Gly(274) --> Ala for the partial reaction steps of the Na(+),K(+)-ATPase transport cycle were analyzed. The mutated residues are part of the long loop ("A-M3 linker") connecting the cytoplasmic A-domain with transmembrane segment M3. It was found that mutation Ile(265) --> Ala displaces the E(1)-E(2) and E(1)P-E(2)P equilibria in favor of E(1)/E(1)P, whereas mutations Thr(267) --> Ala, Gly(271) --> Ala, and Gly(274) --> Ala displace these conformational equilibria in favor of E(2)/E(2)P. The mutations affect both the rearrangement of the cytoplasmic domains (seen by changes in phosphoenzyme properties and apparent ATP/vanadate affinities) and the membrane sector (indicated by change in K(+)/Rb(+) deocclusion rate). Destabilization of E(2)/E(2)P in Ile(265) --> Ala, as well as a direct effect on the intrinsic affinity of the E(2) form for vanadate, may be explained on the basis of the E(2) crystal structures of the Ca(2+)-ATPase, showing interaction of the equivalent isoleucine with conserved residues near the catalytic region of the P-domain. The rate of phosphorylation from ATP was unaffected in Ile(265) --> Ala, indicating a lack of interference with the catalytic function in E(1)/E(1)P. The effects of mutations Thr(267) --> Ala, Gly(271) --> Ala, and Gly(274) --> Ala provide the first evidence in the literature of a relative stabilization of E(2)/E(2)P resulting from perturbation of the A-M3 linker region. These mutations may lead to increased strain of the A-M3 linker in E(1)/E(1)P, increased stability of the A3 helix of the A-M3 linker in E(2)/E(2)P, and/or a change of the orientation of the A3 helix, facilitating its interaction with the P-domain.
Highlights
The Naϩ,Kϩ-ATPase,1 located in the cell membrane, utilizes the free energy derived from the hydrolysis of ATP to import 2 Kϩ in exchange for export of three Naϩ out of the cell [1]
The effects of mutations Thr267 3 Ala, Gly271 3 Ala, and Gly274 3 Ala provide the first evidence in the literature of a relative stabilization of E2/E2P resulting from perturbation of the A-M3 linker region
Comparing the atomic structures of the Ca2ϩ-ATPase in Ca2ϩ-bound E1 forms (E1(Ca2)) [11] and E1(Ca2)1⁄7Mg1⁄7AlF41⁄7ADP [12]) with those of the Ca2ϩ-free E2 forms stabilized by thapsigargin (E2(TG)) [13] and E2(TG)1⁄7Mg1⁄7MgF4 [14]), it appears that the cytoplasmic domains undergo large rearrangements in relation to the E1-E2 conformational changes, and a key event in active transport may be the extensive rotation of the A-domain parallel to the membrane
Summary
Naϩ,Kϩ-ATPase, the Naϩ- and Kϩtransporting adenosine triphosphatase (EC 3.6.1.37); E1 and E2, conformational states of the Naϩ,Kϩ-ATPase; E1P and E2P, phosphorylated conformational states; K0.5, ligand concentration giving halfmaximum activation or inhibition; M1–M10, putative transmembrane segments numbered from the N-terminal end of the peptide chain; TG, thapsigargin. Comparing the atomic structures of the Ca2ϩ-ATPase in Ca2ϩ-bound E1 forms (E1(Ca2)) [11] and E1(Ca2)1⁄7Mg1⁄7AlF41⁄7ADP [12]) with those of the Ca2ϩ-free E2 forms stabilized by thapsigargin (E2(TG)) [13] and E2(TG)1⁄7Mg1⁄7MgF4 [14]), it appears that the cytoplasmic domains undergo large rearrangements in relation to the E1-E2 conformational changes, and a key event in active transport may be the extensive rotation of the A-domain parallel to the membrane. Long before the appearance of the Ca2ϩ-ATPase crystal structures, proteolytic cleavage experiments with Naϩ,KϩATPase had demonstrated that cleavage sites in the cytoplasmic extension linking M3 to the A-domain (“A-M3 linker”) are alternately exposed in relation to the E1-E2 and E1P-E2P conformational transitions. To examine the functional importance of these residues in Naϩ,Kϩ-ATPase, we have studied the effects of the mutations on the overall and partial reactions of the enzyme, using steady-state and transient kinetic measurements
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