title: Probing the conformation of NhaA, a Na+/H+ antiporter from Escherichia coli, with trypsin.
abstract: One of the most striking features of NhaA, an Escherichia coli Na+/H+ antiporter, is its extreme sensitivity to pH. The activity of NhaA increases 2000-fold between pH 6.5 and 8.5. In this work, we investigated whether the activation of NhaA by pH is accompanied by conformational changes which can be detected using trypsin as a probe. We have found that NhaA is susceptible to proteolytic digestion at the pH range where it is activated, suggesting that these two events may be related; at alkaline pH, the protein becomes active and adopts an "open" conformational state in which more domains are exposed to the enzyme. This idea was further supported by results from two mutants of NhaA in which His-225, a residue involved in pH sensing, has been replaced by either Arg or Asp. The mutant H225R is activated at more acidic pH values, while H225D at more alkaline pH. In accordance with the results described for the wild-type protein, H225R was susceptible to digestion by trypsin at the pH at which it undergoes main activation. NhaA has many potential tryptic cleavage sites. However, analysis of the tryptic digestion fragments suggests that at alkaline pH, the protein is exposed to cleavage mainly at hydrophilic loops 6, 7, and 8. Thus, upon activation, NhaA appears to undergo a change in conformation that is reflected in specific regions of the protein.
authors.affiliation: Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Israel.
authors.forename: A
authors.initials: A
authors.lastname: Rothman
authors.forename: Y
authors.initials: Y
authors.lastname: Gerchman
authors.forename: E
authors.initials: E
authors.lastname: Padan
authors.forename: S
authors.initials: S
authors.lastname: Schuldiner
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C102839:NhaA protein, E coli; D010446:Peptide Fragments; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; D014357:Trypsin