Revealing the ligand binding site of NhaA Na+/H+ antiporter and its pH dependence

Abstract

pH and Na(+) homeostasis in all cells requires Na(+)/H(+) antiporters. In most cases, their activity is tightly pH-regulated. NhaA, the main antiporter of Escherichia coli, has homologues in all biological kingdoms. The crystal structure of NhaA provided insights into the mechanism of action and pH regulation of an antiporter. However, the active site of NhaA remained elusive because neither Na(+) nor Li(+), the NhaA ligands, were observed in the structure. Using isothermal titration calorimetry, we show that purified NhaA binds Li(+) in detergent micelles. This interaction is driven by an increase in enthalpy (ΔH of -8000 ± 300 cal/mol and ΔS of -15.2 cal/mol/degree at 283 K), involves a single binding site per NhaA molecule, and is highly specific and drastically dependent on pH; Li(+) binding was observed only at pH 8.5. Combining mutational analysis with the isothermal titration calorimetry measurements revealed that Asp-163, Asp-164, Thr-132, and Asp-133 form the Li(+) binding site, whereas Lys-300 plays an important role in pH regulation of the antiporter.

FIGURE 1.

The crystal structure of the ligand binding site of NhaA. The NhaA residues participating in the NhaA cation binding site are shown (ball and stick) on the respective helices using PyMOL. A, membrane view; B, cytoplasmic view; C, TMs IV/XI assembly. Helices of the assembly and other helices are shown as cylinders. The partial charges of the N and C termini of the short helices (IVc/IVp and XIc/XIp) are indicated.

FIGURE 2.

The Li⁺/H⁺ antiporter activity of WT NhaA and variants K300H and K300R in isolated membrane vesicles. The Li⁺/H⁺ antiporter activity was determined in everted membrane vesicles isolated from EP432 cells expressing WT NhaA or the indicated variants grown in LBK (pH 7.0). The ΔpH across the membranes was monitored using acridine orange, a fluorescence probe of ΔpH. The reaction mixture (2.5 ml) contained 50–100 μg of membrane protein, 0.5 μm acridine orange, 150 mm KCl, 50 mm BTP, 5 mm MgCl₂, and the pH was titrated with HCl as indicated on the figure. The data of typical experiments are shown. At the onset of the reaction, d-lactate (2 mm) was added (↓), and the fluorescence quenching was recorded until a steady-state level of ΔpH (100% quenching) was reached. LiCl (10 mm) was then added (↑), and the new steady state of fluorescence obtained (dequenching) was monitored. Fluorescence dequenching indicated that protons are exiting the vesicles in response to Na⁺ or Li⁺ influx via the antiporter. All experiments were repeated at least three times with practically identical results. AU, arbitrary units.

FIGURE 3.

pH dependence of the Na⁺/Li⁺/H⁺ antiporter activity of NhaA variants H300H and K300R. The antiporter activities of WT (△) and variants K300H (○) and K300R (□) were measured in inverted membrane vesicles as in Fig. 2 at the indicated pH values in the presence of 10 (full symbols) or 100 mm (open symbols) of the indicated cations. The results are expressed as end level of dequenching (in percentage). All experiments were repeated at least three times with practically identical results.

FIGURE 4.

NhaA binds specifically to Li⁺ in a pH-dependent manner. ITC was conducted with WT NhaA purified in DDM and prepared for ITC as described under ”Experimental Procedures.“ The reaction mixture of 300 μl contained wild type NhaA protein (30–40 μm), 0.04% DDM, 50 mm BTP, 150 mm choline chloride, 5 mm MgCl₂, 10% sucrose at the indicated pHs. The injection steps were 2 μl every 200 s. Top panels show the heat change during injection of 40 mm LiCl into a reaction mixture containing 40 μm NhaA at pH 8.5 (A), 35 μm NhaA at pH 8.0 (C), or 36 μm NhaA at pH 9.0 (D). 40 mm KCl was injected into a reaction mixture containing 35 μm NhaA at pH 8.5 (B). Bottom panels display the cumulative heat of reaction as a function of the molar ratio between the protein and the titrant.

FIGURE 5.

Li⁺ binding of NhaA variants with mutations in the cation active site. The experimental procedure was as described in the legend for Fig. 4. The injection was with 40 mm LiCl into a reaction mixture containing 46 μm variant D133C (A) or 48 μm variant D163C (B).