Neutralization of the charge on Asp 369 of Na+,K+-ATPase triggers E1 <--> E2 conformational changes.
This work investigates the role of charge of the phosphorylated aspartate, Asp(369), of Na(+),K(+)-ATPase on E(1) <--> E(2) conformational changes. Wild type (porcine alpha(1)/His(10)-beta(1)), D369N/D369A/D369E, and T212A mutants were expressed in Pichia pastoris, labeled with fluorescein 5'-isothiocyanate (FITC), and purified. Conformational changes of wild type and mutant proteins were analyzed using fluorescein fluorescence (Karlish, S. J. (1980) J. Bioenerg. Biomembr. 12, 111-136). One central finding is that the D369N/D369A mutants are strongly stabilized in E(2) compared with wild type and D369E or T212A mutants. Stabilization of E(2)(Rb) is detected by a reduced K(0.5)Rb for the Rb(+)-induced E(1) <--> E(2)(2Rb) transition. The mechanism involves a greatly reduced rate of E(2)(2Rb) --> E(1)Na with no effect on E(1) --> E(2)(2Rb). Lowering the pH from 7.5 to 5.5 strongly stabilizes wild type in E(2) but affects the D369N mutant only weakly. Thus, this "Bohr" effect of pH on E(1) <--> E(2) is due largely to protonation of Asp(369). Two novel effects of phosphate and vanadate were observed with the D369N/D369A mutants as follows. (a) E(1) --> E(2).P is induced by phosphate without Mg(2+) ions by contrast with wild type, which requires Mg(2+). (b) Both phosphate and vanadate induce rapid E(1) --> E(2) transitions compared with slow rates for the wild type. With reference to crystal structures of Ca(2+)-ATPase and Na(+),K(+)-ATPase, negatively charged Asp(369) favors disengagement of the A domain from N and P domains (E(1)), whereas the neutral D369N/D369A mutants favor association of the A domain (TGES sequence) with P and N domains (E(2)). Changes in charge interactions of Asp(369) may play an important role in triggering E(1)P(3Na) <--> E(2)P and E(2)(2K) --> E(1)Na transitions in native Na(+),K(+)-ATPase.
Belogus
Talya
T
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Haviv
Haim
H
Karlish
Steven J D
SJ
D000818:Animals; D007700:Kinetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013552:Swine
D011994:Recombinant Proteins; D000254:Sodium-Potassium-Exchanging ATPase