Abstract
A single Cys replacement of Glu at position 252 (E252C) in loop VIII-IX of NhaA increases drastically the Km for Na(+) (50-fold) of the Na(+)/H(+) antiporter activity of NhaA and shifts the pH dependence of NhaA activity, by one pH unit, to the alkaline range. In parallel, E252C causes a similar alkaline pH shift to the pH-induced conformational change of loop VIII-IX. Thus, although both the Na(+)/H(+) antiporter activity of wild type NhaA and its accessibility to trypsin at position Lys(249) in loop VIII-IX increase with pH between pH 6.5 and 7.5, the response of E252C occurs above pH 8. Furthermore, probing accessibility of pure E252C protein in dodecyl maltoside solution to 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid revealed that E252C itself undergoes a pH-dependent conformational change, similar to position Lys(249), and the rate of the pH-induced conformational change is increased specifically by the presence of Na(+) or Li(+), the specific ligands of the antiporter. Chemical modification of E252C by N-ethylmaleimide, 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid; [2-(trimethylammonium)ethyl]methane thiosulfonate, or (2-sulfonatoethyl)methanethiosulfonate reversed, to a great extent, the pH shift conferred by E252C but had no effect on the K(m) of the mutant antiporter.
Highlights
Escherichia coli has two antiporters, NhaA [4] and NhaB [5], which exchange Naϩ or Liϩ for Hϩ
E252C causes a similar alkaline pH shift to the pH-induced conformational change of loop VIII–IX. Both the Na؉/H؉ antiporter activity of wild type NhaA and its accessibility to trypsin at position Lys249 in loop VIII–IX increase with pH between pH 6.5 and 7.5, the response of E252C occurs above pH 8
We have identified a novel mutation, E252C in loop VIII–IX, that has a very drastic effect on the pH response of NhaA
Summary
Eco72I BstXI mutant both in the membrane and after solubilization and purification of the protein in DM. E241C and the other two mutations in loop VIII–IX that induce an acidic shift in the pH profile of activity of NhaA, the single amino acid change, E252C, causes a dramatic alkaline shift in the pH dependence of the Naϩ/Hϩ antiporter activity of NhaA and increases drastically the Km of the antiporter to both Naϩ and Liϩ. The E252C mutation causes a similar alkaline shift in the pH dependence of the conformational change in loop VIII–IX. This conformational change at position E252C was found sensitive to both Naϩ and Liϩ, the specific ligands of the NhaA antiporter
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