Proximity of periplasmic loops in the metal-Tetracycline/H(+) antiporter of Escherichia coli observed on site-directed chemical cross-linking.
Our previous study on second-site suppressor mutations of the Tn10-encoded metal-tetracycline/H(+) antiporter suggested that Leu(30) and Ala(354), located in periplasmic loop 1-2 and 11-12, respectively, are conformationally linked to each other (Kawabe, T., and Yamaguchi, A. (1999) FEBS Lett. 457, 169-173). To determine the spatial proximity of these two residues, cross-linking gel-shift assays of the L30C/A354C double mutant were performed after the mutant had been oxidized with Cu(2+)/o-phenanthroline. The results indicated that Leu(30) and Ala(354) are close to each other but that Gly(62), which is located in cytoplasmic loop 2-3, and Ala(354) are distant from each other, as a negative control. Then, a single Cys residue was introduced into each of the six periplasmic loop regions (P1-P6), and eleven double mutants were constructed. Of these eleven double Cys mutants, the L30C/A354C and L30C/T235C mutants showed a mobility shift on oxidation, indicating that P1 is spatially close to P4 as well as P6. In contrast, the other nine mutants, L30C/S92C, L30C/S156C, L30C/S296C, S92C/S296C, S92C/T235C, S92C/A354C, S156C/T235C, S156C/S296C, and S156C/A354C, showed no mobility shift under oxidized conditions on intramolecular cross-linking. The S92C and S296C mutants showed dimerization on intermolecular cross-linking, indicating that P2 and P5 are located at the periphery of the helix bundle.
Kubo
Y
Y
Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan.
Konishi
S
S
Kawabe
T
T
Nada
S
S
Yamaguchi
A
A
D000595:Amino Acid Sequence; D017920:Antiporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001692:Biological Transport; D003300:Copper / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D006859:Hydrogen / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019897:Periplasm / Q000737:chemistry*; D010618:Phenanthrolines / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013752:Tetracycline / Q000737:chemistry*; D013753:Tetracycline Resistance / Q000235:genetics
D017920:Antiporters; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D010618:Phenanthrolines; D013438:Sulfhydryl Compounds; C086977:TETP protein, Clostridium perfringens; C079051:tetA protein, Bacteria; D003300:Copper; D006859:Hydrogen; D013752:Tetracycline; D003545:Cysteine