Unraveling functional and structural interactions between transmembrane domains IV and XI of NhaA Na+/H+ antiporter of Escherichia coli.
A functionally important, interface domain between transmembrane segments (TMSs) IV and XI of the NhaA Na+/H+ antiporter of Escherichia coli has been unraveled. Scanning by single Cys replacements identified new mutations (F136C, G125C, and A137C) that cluster in one face of TMS IV and increase dramatically the Km of the antiporter. Whereas G125C, in addition, causes a drastic alkaline shift to the pH dependence of the antiporter, G338C alleviates the pH control of NhaA. Scanning by double Cys replacements (21 pairs of one replacement per TMS) identified genetically eight pairs of residues that showed very strong negative complementation. Cross-linking of the double mutants identified six double mutants (T132C/G338C, D133C/G338C, F136C/S342C, T132C/S342C, A137C/S342C, and A137C/G338C) of which pronounced intramolecular cross-linking defined an interface domain between the two TMSs. Remarkably, cross-linking by a short and rigid reagent (N,N'-o-phenylenedimaleimide) revived the Li+/H+ antiport activity, whereas a shorter reagent (1,2-ethanediyl bismethanethiosulfonate) revived both Na+/H+ and Li+/H+ antiporter activities and even the pH response of the dead mutant T132C/G338C. Hence, cross-linking at this position restores an active conformation of NhaA.
Galili
Livnat
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Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel.
Herz
Katia
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Dym
Orly
O
Padan
Etana
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; C102839:NhaA protein, E coli; D017923:Sodium-Hydrogen Exchangers