The monoclonal antibody 1F6 identifies a pH-dependent conformational change in the hydrophilic NH2 terminus of NhaA Na+/H+ antiporter of Escherichia coli

One of the most interesting properties of the NhaA Na+/H+ antiporter of Esc herichia coli is the strong regulation of its activity by pH, This regulati on is accompanied by a conformational change that can be probed by digestio n with trypsin and involves the hydrophilic loop connecting the transmembra ne helices VIII-M, In the present work we show that a monoclonal antibody ( mAb), 1F6, recognizes yet another domain of NhaA in a pH-dependent manner. This antibody binds NhaA at pH 8.5 but not at pH 4.5, whereas two other mAb s bind to NhaA independently of pH, The epitope of mAb 1F6 was located at t he NH2 terminus of NhaA by probing proteolytic fragments in Western blot an alysis and amino acid sequencing. The antibody bound to the peptide HLHR-FF SS, starting at the third amino acid of NhaA. A synthetic peptide with this sequence was shown to bind mAb 1F6 both at acidic and alkaline pH suggesti ng that this peptide is accessible to mAb 1F6 in the native protein only at alkaline pH. Although slightly shifted to acidic pH, the pH profile of the binding of mAb 1F6 to the antiporter is similar to that of both the Na+/H antiporter activity as well as to its sensitivity to trypsin, We thus sugg est that these pH profiles reflect a pH-dependent conformational change, wh ich leads to activation of the antiporter, Indeed, a replacement of Gly-338 by Ser (G338S), which alleviates the pH dependence of both the NhaA activi ty as well as its sensitivity to trypsin, affects in a similar pattern the binding of mAb 1F6 to NhaA. Furthermore, the binding site of mAb 1F6 is inv olved in the functioning of the antiporter as follows: a double Cys replace ment H3C/H5C causes an acidic shift by half a pH unit in the pH dependence of the antiporter; N-ethylmaleimide, which does not inhibit the wild-type p rotein, inhibits H3C/H5C antiporter to an extent similar to that exerted by mAb 1F6.