Metal - Tetracycline / H + Antiporter of Escherichia coli Encoded by Transposon Tn 10 T H E ROLE OF A CONSERVED SEQUENCE MOTIF , GXXXXRXGRR , IN A PUTATIVE CYTOPLASMIC

Abstract

The region including the conserved Sere5-Aspe0 dipeptide in the tetracycline/H+ antiporter (TET) encoded by transposon TnlO is thought to play a gating role (Yamaguchi, A., Ono, N., Akasaka, T., Noumi, T., and Sawai, T. (1990) J. Biol. Chem. 265, 1552515530). The dipeptide is in putative interhelix 10op~-~, 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 loop,-, was replaced. Among 10 residues in putative 10op~-~, the important residues, of which substitution resulted in significant reduction or complete loss of the transport activity, were Glyez, Asp“, Glye9, and Arg70. The defect in the transport activity of the Glye2 and Glya9 substitution mutants corresponded to the steric hindrance by the substituents as to the putative &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 Asp“. A “charge-neutralizing” intra-loop salt bridge between Aspse and Arg70 was not likely because the double mutant in which Aspee and Arg70 were replaced with asparagine and leucine, respectively, showed no transport activity. A triple mutant with only one positive charge at Arg7’ in this loop showed about half the wildtype 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 Mets4 + Cys and Arg7’ + Cys mutants; however, the modification of only the Sera5 + Cys mutant caused significant inhibition of the transport activity, indicating that position 65 is a unique position in the structure of loop,-,.

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Cite this paper

@inproceedings{Yamaguchi2001MetalT, title={Metal - Tetracycline / H + Antiporter of Escherichia coli Encoded by Transposon Tn 10 T H E ROLE OF A CONSERVED SEQUENCE MOTIF , GXXXXRXGRR , IN A PUTATIVE CYTOPLASMIC}, author={Akihito Yamaguchi and Yuichi Someya and Tetsuo Sawai}, year={2001} }