Neutralization of the charge on Asp 369 of Na+,K+-ATPase triggers E1 <--> E2 conformational changes

Abstract

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.

FIGURE 1.

Selective labeling of recombinant Na⁺,K⁺-ATPase by FITC and protection by ATP. FITC-labeled membranes show fluorescein label in the α subunit, with ATP 0–1000 μm. The asterisk indicates a contaminant protein. The fluorescence associated with the α subunit was measured with the Typhoon fluorescence imager. The values at each concentrations of ATP are recorded below the appropriate lane, as a percent of control without ATP. WT, wild type.

FIGURE 2.

Purification of fluorescein-labeled wild type (WT) and mutant Na⁺,K⁺-ATPase. Coomassie-stained gel of purified proteins. The fluorescein fluorescence in the α subunit of the purified complexes was visualized with a UV lamp.

FIGURE 3.

K⁺- and Na⁺-induced E₁ ↔ E₂conformational changes of wild type and mutant Na⁺,K⁺-ATPase. Typical fluorescein fluorescence changes of wild type (WT) and mutant proteins labeled with FITC in optimal conditions. The concentrations of Rb⁺ and Na⁺ were chosen to give maximal responses for each protein. a.u., arbitrary units.

FIGURE 4.

Equilibrium titrations of the E₁ ↔ E₂(2Rb) conformational change. Representative titrations of the E₁ ↔ E₂(Rb) change for wild type (WT) and mutants. The data have been normalized, as described under the “Experimental Procedures,” to permit comparison between the different proteins. Solid lines represent curves fitted to the data and provide the parameters in Table 2 for Rb⁺.

FIGURE 5.

Stopped-flow traces of E₂(2Rb) → E₁Na for wild type and D369N/D369A mutants. Each trace for the wild type (WT) and D369N/D369A mutant proteins represents the average of 5–10 replicates. The curves have been fitted to single exponential functions. Rate constants are shown in Table 3. Wild type, black; D369N, gray; D369A, light gray.

FIGURE 6.

Stopped-flow traces of E₁ → E₂(2Rb) for wild type and D369N/D369A mutants. Each trace for the wild type and D369N/D369A mutant proteins represents the average of 5–10 replicates. Final Rb⁺ concentrations are indicated. The curves have been fitted to single exponential functions. Rate constants are shown in Table 3. Wild type (WT), black; D369N, gray; D369A, light gray. Note that the solution in syringe 1 consists of 20–30 μg of FITC-labeled enzymes, choline chloride, plus RbCl (wild type 20 mm, mutants 4 mm) to a total concentration of 165 mm plus 10 mm Hepes, pH 7.5, and that the solution in syringe 2 consists of 165 mm NaCl plus 10 mm Hepes, pH 7.5.

FIGURE 7.

Rate constants of E₁ → E₂(2×) at different concentrations of the monovalent cation. A, rate constants of E₁ → E₂(2Rb) at 20, 30, and 83 mm RbCl for wild type (WT) and D369N/D369A mutants. The graph depicts the fitted rate constants from the data in Table 3. The values of the rate constants for D369N and D369A are virtually identical and cannot be seen as separate points. Note that the solution in syringe 1 consists of 20–50 μg of FITC-labeled enzymes, 165 mm choline chloride, plus 10 mm Hepes, pH 7.5, and the solution in syringe 2 consists of choline chloride + RbCl to a total concentration of 165 mm plus 10 mm Hepes, pH 7.5. B, comparison of rate constants of E₁ → E₂(2×) at different Rb⁺ or K⁺ concentrations for wild type. The data for K⁺ represents fitted rate constants from an experiments similar to that in Fig. 6 for a range of K⁺ concentrations from 10 to 83 mm. The solid line for K⁺ represents the hyperbolic fit with the parameters k_{E2(K) → E1} 125 ± 15 s⁻¹ and the K_K 39 ± 10.5 mm. The data for Rb⁺ are taken from Table 3.

FIGURE 8.

Equilibrium titrations of E₁ ↔ E₂(Rb) at varying pH. A, wild type; B, D369N. The data have been normalized as described under “Experimental Procedures.” Solid lines represent curves fitted to these data and provide the parameters in Table 4. At pH values lower than 7.0, a linear drift was subtracted from the fluorescence signal changes.

FIGURE 9.

Stopped-flow traces of E₂(2Rb) → E₁Na for wild type at pH 7. 5 and 6. Conditions were as under “Experimental Procedures,” except that solutions at pH 6.0 contained 10 mm Mes. Each trace represents the average of 5–10 replicates. The rate was measured at pH 6.0 rather than at 5.5 to avoid a fluorescence drift that interferes with the measurement.

FIGURE 10.

Phosphate- and vanadate-induced E₁ ↔ E₂ conformational changes of wild type (WT) and D369N/D369A mutants.

FIGURE 11.

Stopped-flow traces of rates of phosphate- and vanadate-induced E₁ → E₂ conformational changes for wild type and D369N/D369A mutants. A, phosphate: wild type (WT) and D369N, 25 mm phosphate (Tris), 4 mm MgCl₂, D369A, 25 mm phosphate (Tris). B, vanadate: wild type and mutants 1 mm vanadate (Tris), 4 mm MgCl₂. Traces represent averages of 4–8 replicates and are fitted to a double exponential function; see Table 5.

FIGURE 12.

Active site of Na⁺,K⁺-ATPase in E₂·P conformation and Ca²⁺-ATPase in the E₁ATP conformation. A, active site of Na⁺,K⁺-ATPase in E₂·MgF₄²⁻. Mg²⁺ conformation (PDB code 2XZE). B, as in A without the bound MgF₄²⁻·Mg²⁺. Arrows indicate Asp³⁶⁹ and Glu²¹⁴ and Arg⁵⁴⁴–Glu²¹⁶ and Arg⁶⁸⁵–Glu²³¹ pairs. C, active site of Ca²⁺-ATPase in E₁·AMPPCP (PDB code 1VFP) showing proximity of Arg⁵⁶⁰ and Arg⁶⁷⁸ to the bound AMPPCP. The figure was drawn with PyMOL. P domain, green; A domain, red; N domain, blue.

FIGURE 13.

Scheme showing alternative pathways for conformational change from E₁ to E₂P.