Abstract
The Na(+)/H(+) exchanger isoform 1 is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammals. We characterized the structural and functional aspects of the critical transmembrane (TM) segment IV. Each residue was mutated to cysteine in cysteine-less NHE1. TM IV was exquisitely sensitive to mutation with 10 of 23 mutations causing greatly reduced expression and/or activity. The Phe(161) --> Cys mutant was inhibited by treatment with the water-soluble sulfhydryl-reactive compounds [2-(trimethylammonium)ethyl]methanethiosulfonate and [2-sulfonatoethyl]methanethiosulfonate, suggesting it is a pore-lining residue. The structure of purified TM IV peptide was determined using high resolution NMR in a CD(3)OH:CDCl(3):H(2)O mixture and in Me(2)SO. In CD(3)OH: CDCl(3):H(2)O, TM IV was structured but not as a canonical alpha-helix. Residues Asp(159)-Leu(162) were a series of beta-turns; residues Leu(165)-Pro(168) showed an extended structure, and residues Ile(169)-Phe(176) were helical in character. These three structured regions rotated quite freely with respect to the others. In Me(2)SO, the structure was much less defined. Our results demonstrate that TM IV is an unusually structured transmembrane segment that is exquisitely sensitive to mutagenesis and that Phe(161) is a pore-lining residue.
Highlights
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
To examine which amino acids of TM IV—Transmembrane segment four (TM IV) were critical for the activity of the Naϩ/Hϩ exchanger and which amino acids were pore-lining, we used the cysteine-less Naϩ/Hϩ exchanger
Each residue in TM IV of cNHE1 was individually mutated to a cysteine residue
Summary
Materials—15N-Labeled ammonium sulfate and all deuterated NMR solvents were from Cambridge Isotope Laboratories (Andover, MA). To test the effect of MTSET and MTSES on activity of the NHE1 mutants, we used the standard Naϩ/Hϩ exchanger assay with ammonium chloride-induced acidification of the cells. In this case cells were acidified two times as described above. By using uniformly 15N-labeled peptide, the three-dimensional 15N-edited NOESY-HSQC (150-ms mixture), TOCSY-HSQC (57-ms mixture, DIPSI spin lock), and HNHA experiments were acquired at 500 MHz on a Varian Inova spectrometer. After all major NOE violations were resolved, the three-bond J-coupling values were incorporated directly into the simulated annealing protocol [21] using Karplus coefficients as specified by Vuister and Bax [19] for the HNHA experiment for the more structured CD3OH: CDCl3:H2O condition. The final sets of restraints have been deposited in the Protein Data Bank (entry 1Y4E) along with the 100 lowest energy structures for the CD3OH:CDCl3:H2O solvent condition
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
