Roles of Conserved Arginine Residues in the Metal−Tetracycline/H⁺ Antiporter of Escherichia coli

Abstract

Seven arginine residues are conserved in all the tetracycline/H+ antiporters of Gram-negative bacteria. Four (Arg67, -70, -71, and -127) of them are located in the putative cytoplasmic loop regions and three (Arg31, -101, and -238) in the putative periplasmic loop regions [Eckert, B., and Beck, C. F. (1989) J. Biol. Chem. 264, 11663-11670]. These arginine residues were replaced by alanine, lysine, or cysteine one by one through site-directed mutagenesis. None of the mutants showed significant alteration of the protein expression level. The mutants resulting in the replacement of Arg31, Arg67, Arg71, and Arg238 with either Ala, Cys, or Lys retained tetracycline resistance levels comparable to that of the wild type. Among them, only the Arg238 --> Ala mutant showed very low transport activity in everted membrane vesicles, probably due to the instability of the mutant protein. The replacement of Arg70 and Arg127 with Ala or Cys resulted in a drastic decrease in the drug resistance and almost complete loss of the transport activity, while the Lys replacement mutants retained significant resistance and transport activity, indicating that the positively charged side chains at these positions conferred the transport function. On the other hand, neither the Ala, Cys, nor Lys replacement mutant of Arg101 exhibited any drug resistance or transport activity. As for the reactivity of the Cys replacement mutants, only two (Arg71 --> Cys and Arg101 --> Cys) were not reactive with NEM, the other five mutants being highly or moderately reactive. The reactivity of the cysteine-scanning mutants around Arg101 with NEM revealed that Arg101 is located in transmembrane helix IV. It is not likely that Arg101 confers the protein folding through a salt bridge with a transmembrane acidic residue because no double mutants involving Arg101 --> Ala and the replacement of one of three transmembrane acidic residues (Asp15, Asp84, and Asp285) showed the recovery of any tetracycline resistance or transport activity. The effect of tetracycline on the [14C]NEM binding to the combined mutants S65C/R101A and L97C/R101A suggests that Arg101 may cause a substrate-induced conformational change of the putative exit gate of TetA(B).

Cysteine-Scanning Mutagenesis of the Periplasmic Loop Regions of PomA, a Putative Channel Component of the Sodium-Driven Flagellar Motor in Vibrio alginolyticus

“…Cysteine or alanine scanning is known as a useful method for the functional analysis of various ion channels and transporters (14,23,27,35). Applying this method to investigate the flagellar motor was expected to give us information not only about functional residues but also about the structural changes around each residue from the binding effects of SH-modifying reagents.…”

Identification of structural and functional domains of the tetracycline efflux protein TetA(P) from Clostridium perfringens

“…In addition, we note that this random mutagenesis approach produced an unexpectedly high percentage (42 %) of mutant TetA(P) derivatives from which no detectable TetA(P) protein was observed, each resulting in tetracycline sensitivity (MIC 2 µg ml −" ). Previous studies of the TetA(B) protein, which also involved the examination of large numbers of mutant derivatives, reported that very few mutant proteins were unable to be detected with anti-TetA(B) protein antiserum (Kimura et al, 1998 ;Yamaguchi et al, 1992c, d).…”

“…It has been experimentally shown to have 12 TMDs (Allard & Bertrand, 1992 ;Eckert & Beck, 1989 ;Kimura et al, 1997) and has a very high level of amino acid sequence identity to a range of tetracycline efflux proteins from other Gram-negative bacteria. Many residues that are required for TetA(B) activity have been identified (Kimura et al, 1998 ;Yamaguchi et al, 1990aYamaguchi et al, , 1992bYamaguchi et al, , 1993a. In particular, three negatively charged amino acids, D15, D84 and D285, which are situated within TMDs, are essential for tetracycline transport function, probably by forming a transmembrane efflux pathway for the tetracycline complex (Yamaguchi et al, 1992a).…”

Role of the Charge Interaction between Arg70 and Asp120 in the Tn10-encoded Metal-Tetracycline/H+ Antiporter of Escherichia coli

“…Three motifs, GXXXDRXGRR in loop 2-3, DXXXXXXR in loop 4 -5, and PESPR in loop 6 -7, are also found in the cytoplasmic face of the N-terminal half of TetA(B). We have already focused on the loop 2-3 motif itself (11) and on several charged residues in these motifs (12)(13)(14)(15), and we found Asp 66 and Arg 70 in loop 2-3, Asp 120 and Arg 127 in loop 4 -5, and Glu 181 in loop 6 -7 to be residues essential or important for function, which was based on the loss of transport activity and altered kinetic constants. However, we have not yet identified the precise role(s) of these residues, except that the negative charge of Asp 66 may first interact with a monocationic substrate, tetracycline-divalent cation complex (16).…”