A pH-dependent conformational change of NhaA Na(+)/H(+) antiporter of Escherichia coli involves loop VIII-IX, plays a role in the pH response of the protein, and is maintained by the pure protein in dodecyl maltoside.
Digestion with trypsin of purified His-tagged NhaA in a solution of dodecyl maltoside yields two fragments at alkaline pH but only one fragment at acidic pH. Determination of the amino acid sequence of the N terminus of the cleavage products show that the pH-sensitive cleavage site of NhaA, both in isolated everted membrane vesicles as well as in the pure protein in detergent, is Lys-249 in loop VIII-IX, which connects transmembrane segment VIII to IX. Interestingly, the two polypeptide products of the split antiporter remain complexed and co-purify on Ni(2+)-NTA column. Loop VIII-IX has also been found to play a role in the pH regulation of NhaA; three mutations introduced into the loop shift the pH profile of the Na(+)/H(+) antiporter activity as measured in everted membrane vesicles. An insertion mutation introducing Ile-Glu-Gly between residues Lys-249 and Arg-250 (K249-IEG-R250) and Cys replacement of either Val-254 (V254C) or Glu-241 (E241C) cause acidic shift of the pH profile of the antiporter by 0.5, 1, and 0.3 pH units, respectively. Interestingly, the double mutant E241C/V254C introduces a basic shift of more than 1 pH unit with respect to the single mutation V254C. Taken together these results imply the involvement of loop VIII-IX in the pH-induced conformational change, which leads to activation of NhaA at alkaline pH.
Gerchman
Y
Y
Division of Microbial and Molecular Ecology, Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
Rimon
A
A
Padan
E
E
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D005960:Glucosides / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017421:Sequence Analysis; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000235:genetics; D014357:Trypsin
D029968:Escherichia coli Proteins; D005960:Glucosides; C102839:NhaA protein, E coli; D010446:Peptide Fragments; D017923:Sodium-Hydrogen Exchangers; C040358:dodecyl maltoside; D014357:Trypsin