title: Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10. The role of a conserved sequence motif, GXXXXRXGRR, in a putative cytoplasmic loop between helices 2 and 3.
abstract: The region including the conserved Ser65-Asp66 dipeptide in the tetracycline/H+ antiporter (TET) encoded by transposon Tn10 is thought to play a gating role (Yamaguchi, A., Ono, N., Akasaka, T., Noumi, T., and Sawai, T. (1990) J. Biol. Chem. 265, 15525-15530). The dipeptide is in putative interhelix loop2-3, which also includes the conserved sequence motif, GXXXXRXGRR, found in all TET proteins and sugar/H+ symporters. Through the combination of localized random and site-directed mutagenesis, each residue in loop2-3 was replaced. Among 10 residues in putative loop2-3, the important residues, of which substitution resulted in significant reduction or complete loss of the transport activity, were Gly62, Asp66, Gly69, and Arg70. The defect in the transport activity of the Gly62 and Gly69 substitution mutants corresponded to the steric hindrance by the substituents as to the putative beta-turn structure of the peptide backbone containing these glycines. Of 3 conserved Arg residues, the replacement of only Arg70 caused complete loss of the activity except for replacement with Lys, indicating the importance of a positive charge at this position, which is similar to the essentiality of a negative charge at Asp66. A "charge-neutralizing" intra-loop salt bridge between Asp66 and Arg70 was not likely because the double mutant in which Asp66 and Arg70 were replaced with asparagine and leucine, respectively, showed no transport activity. A triple mutant with only one positive charge at Arg70 in this loop showed about half the wild-type activity, indicating that the polycationic nature of the loop was not critical for the activity. Cys mutants as to the unessential residues in the loop were modifiable with N-ethylmaleimide, except for the Met64----Cys and Arg71----Cys mutants; however, the modification of only the Ser65----Cys mutant caused significant inhibition of the transport activity, indicating that position 65 is a unique position in the structure of loop2-3.
authors.affiliation: Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Yamaguchi
authors.forename: Y
authors.initials: Y
authors.lastname: Someya
authors.forename: T
authors.initials: T
authors.lastname: Sawai
mesh_terms: D000595:Amino Acid Sequence; D017920:Antiporters; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005033:Ethylmaleimide / Q000737:chemistry; D005798:Genes, Bacterial; D008670:Metals; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics; D013329:Structure-Activity Relationship; D013752:Tetracycline / Q000378:metabolism*; D013753:Tetracycline Resistance
chemical_list: D017920:Antiporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004251:DNA Transposable Elements; D008670:Metals; D009838:Oligodeoxyribonucleotides; D012097:Repressor Proteins; C079051:tetA protein, Bacteria; D013752:Tetracycline; D005033:Ethylmaleimide