Here, we used isothermal titration calorimetry (ITC) combined with mutagenesis and biochemical analysis to explore the binding of Na+/Li+ to wild type NhaA and NhaA variants purified in detergent micelles. We revealed the thermodynamics of Li+ binding to NhaA; the high specificity, stoichiometry (1Li+/NhaA), and drastic pH dependence of binding; and that Asp-163, Asp-164, Thr-132, and Asp-133 form the binding site, whereas Lys-300 plays a major role in the pH regulation of NhaA.

Therefore, the question arises whether the pH dependence of activity is a reflection of the pH dependence of the ligand binding to NhaA or a reaction following binding. To answer this question, we measured the pH dependence of Li+ binding to NhaA in different ITC reaction buffers with pH values of 6.5, 7.0, 7.5 (not shown), 8.0, 8.5, and 9.0 (Fig. 4, A, C, and D). However, binding was observed only at pH 8.5 (Fig. 4A). These results imply that ligand binding to NhaA is extremely sensitive to pH.

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Unfortunately, we could not test K300H because the protein aggregated. K300R gave just a hint of binding only at pH 9.0, but no quantitative analysis could be done. This is not surprising because of the very high Km of K300R (Table 1). Taken together, we conclude that the Li+ binding site of NhaA is formed by Asp-163, Asp-164, Asp-133, and Thr-132 and that Lys-300 dramatically affects the pH response of NhaA in addition to being involved in its antiporter activity.