Biomaterial Database

Roles of conserved arginine residues in the metal-tetracycline/H+ antiporter of Escherichia coli. 1998

T Kimura, and M Nakatani, and T Kawabe, and A Yamaguchi

Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Japan.

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).

UI	MeSH Term	Description	Entries
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D011485	Protein Binding	The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments.	Plasma Protein Binding Capacity,Binding, Protein
D002250	Carbon Radioisotopes	Unstable isotopes of carbon that decay or disintegrate emitting radiation. C atoms with atomic weights 10, 11, and 14-16 are radioactive carbon isotopes.	Radioisotopes, Carbon
D002462	Cell Membrane	The lipid- and protein-containing, selectively permeable membrane that surrounds the cytoplasm in prokaryotic and eukaryotic cells.	Plasma Membrane,Cytoplasmic Membrane,Cell Membranes,Cytoplasmic Membranes,Membrane, Cell,Membrane, Cytoplasmic,Membrane, Plasma,Membranes, Cell,Membranes, Cytoplasmic,Membranes, Plasma,Plasma Membranes
D003545	Cysteine	A thiol-containing non-essential amino acid that is oxidized to form CYSTINE.	Cysteine Hydrochloride,Half-Cystine,L-Cysteine,Zinc Cysteinate,Half Cystine,L Cysteine
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005033	Ethylmaleimide	A sulfhydryl reagent that is widely used in experimental biochemical studies.	N-Ethylmaleimide,N Ethylmaleimide
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D001120	Arginine	An essential amino acid that is physiologically active in the L-form.	Arginine Hydrochloride,Arginine, L-Isomer,DL-Arginine Acetate, Monohydrate,L-Arginine,Arginine, L Isomer,DL Arginine Acetate, Monohydrate,Hydrochloride, Arginine,L Arginine,L-Isomer Arginine,Monohydrate DL-Arginine Acetate
D001224	Aspartic Acid	One of the non-essential amino acids commonly occurring in the L-form. It is found in animals and plants, especially in sugar cane and sugar beets. It may be a neurotransmitter.	(+-)-Aspartic Acid,(R,S)-Aspartic Acid,Ammonium Aspartate,Aspartate,Aspartate Magnesium Hydrochloride,Aspartic Acid, Ammonium Salt,Aspartic Acid, Calcium Salt,Aspartic Acid, Dipotassium Salt,Aspartic Acid, Disodium Salt,Aspartic Acid, Hydrobromide,Aspartic Acid, Hydrochloride,Aspartic Acid, Magnesium (1:1) Salt, Hydrochloride, Trihydrate,Aspartic Acid, Magnesium (2:1) Salt,Aspartic Acid, Magnesium-Potassium (2:1:2) Salt,Aspartic Acid, Monopotassium Salt,Aspartic Acid, Monosodium Salt,Aspartic Acid, Potassium Salt,Aspartic Acid, Sodium Salt,Calcium Aspartate,Dipotassium Aspartate,Disodium Aspartate,L-Aspartate,L-Aspartic Acid,Magnesiocard,Magnesium Aspartate,Mg-5-Longoral,Monopotassium Aspartate,Monosodium Aspartate,Potassium Aspartate,Sodium Aspartate,Aspartate, Ammonium,Aspartate, Calcium,Aspartate, Dipotassium,Aspartate, Disodium,Aspartate, Magnesium,Aspartate, Monopotassium,Aspartate, Monosodium,Aspartate, Potassium,Aspartate, Sodium,L Aspartate,L Aspartic Acid