Fe(2+)-tetracycline-mediated cleavage of the Tn10 tetracycline efflux protein TetA reveals a substrate binding site near glutamine 225 in transmembrane helix 7.
TetA specified by Tn10 is a class B member of a group of related bacterial transport proteins of 12 transmembrane alpha helices that mediate resistance to the antibiotic tetracycline. A tetracycline-divalent metal cation complex is expelled from the cell in exchange for a entering proton. The site(s) where tetracycline binds to this export pump is not known. We found that, when chelated to tetracycline, Fe(2+) cleaved the backbone of TetA predominantly at a single position, glutamine 225 in transmembrane helix 7. The related class D TetA protein from plasmid RA1 was cut at exactly the same position. There was no cleavage with glycylcycline, an analog of tetracycline that does not bind to TetA. The Fe(2+)-tetracycline complex was not detectably transported by TetA. However, cleavage products of the same size as with Fe(2+) occurred with Co(2+), known to be cotransported with tetracycline. The known substrate Mg (2+)-tetracycline interfered with cleavage by Fe(2+). These findings suggest that cleavage results from binding at a substrate-specific site. Fe(2+) is known to be able to cleave amide bonds in proteins at distances up to approximately 12 A. We conclude that the alpha carbon of glutamine 225 is probably within 12 A of the position of the Fe(2+) ion in the Fe(2+)-tetracycline complex bound to the protein.
Center for Adaptation Genetics and Drug Resistance and Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA. laura.mcmurry@tufts.edu
Laura M
LM
McMurry
Mila L
ML
Aldema-Ramos
Stuart B
SB
Levy
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D017920:Antiporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D001692:Biological Transport; D004251:DNA Transposable Elements / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005296:Ferrous Compounds / Q000378:metabolism*; D005973:Glutamine / Q000737:chemistry; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009154:Mutation; D013379:Substrate Specificity; D013752:Tetracycline / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D017920:Antiporters; D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D005296:Ferrous Compounds; C079051:tetA protein, Bacteria; D005973:Glutamine; D013752:Tetracycline