PMID:15039449
| Citation |
Galili, L, Herz, K, Dym, O and Padan, E (2004) Unraveling functional and structural interactions between transmembrane domains IV and XI of NhaA Na+/H+ antiporter of Escherichia coli. J. Biol. Chem. 279:23104-13 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1074/jbc.M400288200 |
| Keywords |
Amino Acid Sequence; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Cross-Linking Reagents; Escherichia coli/metabolism; Escherichia coli Proteins; Models, Molecular; Molecular Sequence Data; Protein Conformation; Protein Structure, Tertiary; Sodium-Hydrogen Antiporter/chemistry; Sodium-Hydrogen Antiporter/genetics; Sodium-Hydrogen Antiporter/metabolism; Structure-Activity Relationship |
| edit table |
Significance
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Annotations
<annotationlinks/>
EcoliWiki Links
Add links to pages that link here (e.g. gene, product, method pages)
References
See Help:References for how to manage references in EcoliWiki.