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

Abstract

A single Cys replacement of Glu at position 252 (E252C) in loop VIII-IX of NhaA increases drastically the K m 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.

Structure-based Functional Study Reveals Multiple Roles of Transmembrane Segment IX and Loop VIII–IX in NhaA Na+/H+ Antiporter of Escherichia coli at Physiological pH

“…Fig. 2B shows H253C as an example together with the pH profile of the activity of E252C, previously shown to be alkaline shifted by 1 pH unit (16). Hence in this screen, Cys replacements of many residues in the N-terminal half of TMS IX and the vicinal part of loop VIII-IX were found to affect the pH response of NhaA in line with the contention that this NhaA segment participates in the pH sensor.…”

“…Notably Cys replacement E252C was shown previously to have an apparent K m for Na ϩ of at least 10 times that of the wild type (Ref. 16 and Table 1). Here we found that Cys replacement L251C (next to Glu 252 ) also increased by 4-fold the apparent K m for Na ϩ of NhaA (Table 1).…”

“…Cys Replacement Mutations, Expression in the Membrane, and Growth Phenotypes at Physiological pH-The crystal structure of the acid pH-locked conformation of NhaA (11) and structure-based computation (33) combined with genetic and biochemical data (16,25) have suggested that the cytoplasmic end of helix IX and the in-tandem part of loop VIII-IX contain amino acid residues involved in the pH sensor of NhaA. To identify these residues, we systematically replaced with Cys the amino acids residues (each separately) in segment Glu 241 -Val 259 of NhaA excluding E241C, V254C (6,25), and E252C (16) that were isolated previously (Fig.…”

“…pCL-BSTX, a pCL-GMAR100 derivative (16) encodes Cys-less NhaA with a silent BstX site at codon 248 in nhaA. pCL-AXH3, a derivative of pCL-AXH2, contains a BstXI silent site at codon 248 in nhaA (17).…”

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

“…The functioning of the protein and its homologues has been a subject of numerous studies during the last decade, as the sodium/proton exchange is crucial in the development of ischemia heart disease and reperfusion (Blatter and McGuigan 1991;Fliegel 1999). While certain functional insights were achieved via biochemical and mutational studies (Gerchman et al 1999;Tzubery et al 2004;Zhang et al 2002), detailed molecular mechanisms of ion transport and regulation of sodium/proton exchange still challenges structural biology. X-ray diffraction on NhaA crystals has recently provided detailed insights into the packing of transmembrane domains (Hunte et al 2005) and, particularly, the architecture of Na + -binding pocket formed by α-helices IV, V and XI (Fig.…”