The Monoclonal Antibody 1F6 Identifies a pH-dependent Conformational Change in the Hydrophilic NH2 Terminus of NhaA Na+/H+ Antiporter ofEscherichia coli

Abstract

One of the most interesting properties of the NhaA Na ؉ /H ؉ antiporter of Escherichia coli is the strong regulation of its activity by pH. This regulation is accompanied by a conformational change that can be probed by digestion with trypsin and involves the hydrophilic loop connecting the transmembrane helices VIII-IX. 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 mAbs bind to NhaA independently of pH. The epitope of mAb 1F6 was located at the NH 2 terminus of NhaA by probing proteolytic fragments in Western blot analysis and amino acid sequencing. The antibody bound to the peptide HLHR-FFSS, 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 suggesting 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 suggest that these pH profiles reflect a pH-dependent conformational change, which leads to activation of the antiporter. Indeed, a replacement of Gly-338 by Ser (G338S), which alleviates the pH dependence of both the NhaA activity 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 involved in the functioning of the antiporter as follows: a double Cys replacement 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 protein, inhibits H3C/H5C antiporter to an extent similar to that exerted by mAb 1F6.

Mutation E252C Increases Drastically the K Value for Na+ and Causes an Alkaline Shift of the pH Dependence of NhaA Na+/H+ Antiporter of Escherichia coli

“…In the present work, we have identified a novel mutation, E252C in loop VIII-IX, that has a very drastic effect on the pH response of NhaA. Whereas other mutations in this loop (E241C and V254C (19)) and other loops (15,18) cause acidic shift in the pH dependence of the activity of NhaA, Cys replacement mutation E252C causes an alkaline shift of one pH unit. As yet, such an alkaline shift of the pH response of NhaA has previously been found only in mutations at position 127 in the middle of TMS IV (21).…”

“…Monoclonal antibody 1F6 raised against the NhaA antiporter (17) identified that the N terminus of NhaA, its epitope, responds to pH (18). The antibody binds NhaA at pH 8.5 but not at pH 4.5.…”

“…The antibody binds NhaA at pH 8.5 but not at pH 4.5. Furthermore, H3C/H5C, a double mutation in this domain, changes the pH profile of NhaA (18).…”

Characterizing folding, structure, molecular interactions and ligand gated activation of single sodium/proton antiporters

“…Experiments on cleaved NhaA allowed assign one class to pulling from the Cterminal end and the other one to pulling from the N-terminal end. However, the analysis also showed that the majority of unfolding events reflected pulling the C-terminal end of NhaA; an effect, which may be explained by the different accessibility of both terminal ends to the AFM stylus (Venturi et al 2000).…”

Production and characterization of monoclonal antibodies sensitive to conformation in the 5HT2c serotonin receptor

“…Another example concerns the biochemistry of NhaA, a bacterial Na ϩ /H ϩ antiporter that is strongly regulated by pH (6). A specific mAb was shown able to bind only the alkaline pH state of NhaA (7), and eptiope mapping disclosed a previously unpredicted conformational change upon activation (8).…”