Neutralization of the charge on Asp369 of Na+, K+-ATPase triggers E1 ↔ E2 conformational changes

Abstract

This work investigates the role of charge of the phosphorylated aspartate, Asp369, of Na+,K+-ATPase on E1↔E2 conformational changes. Wild type (porcine α1/His10-β1), D369N/D369A/D369E, and T212A mutants were expressed in Pichia pastoris, labeled with fluorescein 5-pm-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 E2 compared with wild type and D369E or T212A mutants. Stabilization of E2(Rb) is detected by a reduced K0.5Rb for the Rb+- induced E1 ↔ E2(2Rb) transition. The mechanism involves a greatly reduced rate of E2(2Rb)→E1Na with no effect on E1 → E2(2Rb). Lowering the pH from 7.5 to 5.5 strongly stabilizes wild type in E2 but affects the D369N mutant only weakly. Thus, this "Bohr" effect of pH on E1 ↔ E2 is due largely to protonation of Asp369. Two novel effects of phosphate and vanadate were observed with the D369N/ D369A mutants as follows. (a) E1 → E2·P is induced by phosphate without Mg2+ ions by contrast with wild type, which requires Mg2+. (b) Both phosphate and vanadate induce rapid E1→E2 transitions compared with slow rates for the wild type. With reference to crystal structures of Ca2+-ATPase and Na+,K+-ATPase, negatively charged Asp369 favors disengagement of the A domain from N and P domains (E1), whereas the neutral D369N/D369A mutants favor association of the A domain (TGES sequence) with P and N domains (E2). Changes in charge interactions of Asp369 may play an important role in triggering E1P(3Na) ↔ E2P and E2(2K) → E1Na transitions in native Na+,K+-ATPase. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.