Neutralization of the Charge on Asp369 of Na+,K+-ATPase Triggers E1 ↔ E2 Conformational Changes

Abstract

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) 7 E 2 P and E 2 (2K) 3 E 1 Na transitions in native Na ؉ ,K ؉ -ATPase.The kinetic mechanism of P-type cation pumps is now well established. Active cation transport involves covalent phosphorylation by ATP and dephosphorylation of an aspartate residue coupled to cation movements mediated by E 1 7 E 2 2 conformational changes. The molecular mechanism is, of course, the central question of energy transduction. Molecular structures of the sarcoplasmic reticulum Ca 2ϩ -ATPase (SERCA1) in several conformations are available (1-3). Two crystal structures of the Na ؉ ,K ؉ -ATPase, consisting of ␣, ␤, and FYXD subunits (4), in an E 2 (2Rb)-MgF 4 2Ϫ conformation (equivalent to E 2 (2Rb)⅐P) have been published recently (5, 6). The architecture of the ␣ subunit is very similar to the Ca 2ϩ -ATPase, consisting of head, stalk, and membrane sectors, with 10 trans-membrane segments and a cytoplasmic sector consisting of N (nucleotide binding), P (phosphorylating), and A (anchor or actuator) domains. Compared with the first published structure of the Na ϩ ,K ϩ -ATPase at 3.5 Å (5), the more recent structure at 2.4 Å (6) reveals greater detail of the cation binding domain, resolution of the ␤ subunit ectodomain, and an FXYD protein ectodomain.In the case of Na ϩ ,K ϩ -ATPase, three Na ؉ ions and two K ؉ ions are transported in the E 1 ATP 3 E 1 P(3Na) 3E 2 P and the E 2 P 3 E 2 (2K) 3 E 1 ATP halves of the cycle, respectively, at the expense of one molecule of ATP. Binding of ATP with low affinity to E 2 (2K) is the first step in the catalytic cycle and is followed by accelerated conversion of E 2 (2K)ATP to E 1 ⅐ATP in which ATP is bound with high affinity (7-9). In the case of Ca 2ϩ -ATPase, two Ca 2ϩ ions and two protons are transported in the E 1 ATP 3 E 1 P 3 E 2 P E 2 P 3 E 2 3 E 1 ATP halves of the cycle, respectively.The mechanism of energy transduction by P-type cation pumps is best addressed by reference to the Ca 2ϩ -ATPase, which has been crystallized in most of the relevant conformations, reviewed recently in detail (1-3). Crystal structures of Ca 2ϩ -ATPase show mainly rigid body movements of the N, P, and A domains, mediated by the flexible linkers between the N and P and between the A domain and trans-membrane segment (M1-M3), coupled mechanically to movements of M1-M6 trans-membrane segments that allow alternate access of two Ca 2ϩ ions and two protons to the occlusion sites within M4, M5, M6, and M8. M7-M10 segments appear to act as immovable anchors. In the E 1 ATP state, N and P domains are in close proximity cross-linked by the bound ATP, whereas the A domain is displaced to one side. Following phosphorylation to E 1 P, the conformational change to E 2 P involves a large rotation of the A domain bringing it into close proximity with th...

FXYD Proteins Stabilize Na,K-ATPase

“…At short incubation times, a small but significant enhancement of Na ϩ binding and ATP-induced activity was observed. A similar phenomenon was observed previously in measurements of Na,KATPase activity and was attributed to activation of a fraction of dormant pumps by equilibration with the lipid (26,36).…”

“…The proteins were added to a medium containing 150 mM choline chloride; 10 mM Hepes (Tris), pH 7.5. Fluorescence changes indicative of E 1 f E 2 (2Rb) and E 2 (2Rb)fE 1 3Na conformational changes were measured by addition of 20 mM RbCl and then 50 mM NaCl (36). The times of incubation at 45°C in minutes and fluorescence signal amplitudes, ⌬F, are shown in the figure for the control (upper panel) and ϩFXYD1 (lower panel), respectively.…”

“…The experiment in Fig. 6 utilized fluorescein-labeled recombinant ␣1␤1 complex for study of E 1 7 E 2 conformational changes (as described recently (36) and under "Experimental Procedures"). The enzymes used in Fig.…”

“…Membranes expressing human ␣1␤1 were labeled with 1 M FITC at pH 9, and the fluorescein-labeled ␣1␤1 complex was purified as described (36). The fluorescein-labeled ␣1␤1FXYD1 complex was produced by adding FXYD1 at a molar ratio of ϳ10:1 overnight at 4°C.…”

Molecular Mechanisms and Kinetic Effects of FXYD1 and Phosphomimetic Mutants on Purified Human Na,K-ATPase

“…In the absence of ATP, the rate-limiting step of E 2 (2K) 3 E 1 3Na is thought to be dissociation of salt bridges, Glu-216 to Arg-544 between A and N and Glu-231 to Arg-685 between A and P domains (12,50). ATP accelerates the rate of dissociation of these two salt bridges by competing with Glu-216 and Glu-231 for Arg-544 and Arg-685, which are direct ATP binding residues, and thus strongly accelerates the rate of the reaction sequence E 2 (2K) 3 E 1 3Na.…”