Proximity of cytoplasmic and periplasmic loops in NhaA Na+/H+ antiporter of Escherichia coli as determined by site-directed thiol cross-linking.
The unique trypsin cleavable site of NhaA, the Na(+)/H(+) antiporter of Escherichia coli, was exploited to detect a change in mobility of cross-linked products of NhaA by polyacrylamide gel electrophoresis. Double-Cys replacements were introduced into loops, one on each side of the trypsin cleavage site (Lys 249). The proximity of paired Cys residues was assessed by disulfide cross-linking of the two tryptic fragments, using three homobifunctional cross-linking agents: 1,6-bis(maleimido)hexane (BMH), N,N'-o-phenylenedimaleimide (o-PDM), and N,N'-p-phenylenedimaleimide (p-PDM). The interloop cross-linking was found to be very specific, indicating that the loops are not merely random coils that interact randomly. In the periplasmic side of NhaA, two patterns of cross-linking are observed: (a) all three cross-linking reagents cross-link very efficiently between the double-Cys replacements A118C/S286C, N177C/S352C, and H225C/S352C; (b) only BMH cross-links the double-Cys replacements A118C/S352C, N177C/S286C, and H225C/S286C. In the cytoplasmic side of NhaA, three patterns of cross-linking are observed: (a) all three cross-linking reagents cross-link very efficiently the pairs of Cys replacements L4C/E252C, S146C/L316C, S146C/R383C, and E241C/E252C; (b) BMH and p-PDM cross-link efficiently the pairs of Cys replacements S87C/E252C, S87C/L316C, and S146C/E252C; (c) none of the reagents cross-links the double-Cys replacements L4C/L316C, L4C/R383C, S87C/R383C, A202C/E252C, A202C/L316C, A202C/R383C, E241C/L316C, and E241C/R383C. The data reveal that the N-terminus and loop VIII-IX that have previously been shown to change conformation with pH are in close proximity within the NhaA protein. The data also suggest close proximity between N-terminal and C-terminal helices at both the cytoplasmic and the periplasmic face of NhaA.
Rimon
A
A
Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Israel.
Tzubery
T
T
Galili
L
L
Padan
E
E
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D003593:Cytoplasm / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019897:Periplasm / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014407:Tumor Cells, Cultured
D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D005452:Fluoresceins; D005456:Fluorescent Dyes; C102839:NhaA protein, E coli; D010446:Peptide Fragments; D017923:Sodium-Hydrogen Exchangers; D013438:Sulfhydryl Compounds; C037041:fluorescein 5-maleimide; D003545:Cysteine