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Journal of Bacteriology, September 2002, p. 5113-5120, Vol. 184, No. 18
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.18.5113-5120.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Fe²⁺-Tetracycline-Mediated Cleavage of the Tn10 Tetracycline Efflux Protein TetA Reveals a Substrate Binding Site near Glutamine 225 in Transmembrane Helix 7

Laura M. McMurry,^* Mila L. Aldema-Ramos, and Stuart B. Levy

Center for Adaptation Genetics and Drug Resistance and Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111

Received 6 November 2001/ Accepted 17 June 2002

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, Fe²⁺ 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 Fe²⁺-tetracycline complex was not detectably transported by TetA. However, cleavage products of the same size as with Fe²⁺ occurred with Co²⁺, known to be cotransported with tetracycline. The known substrate Mg ²⁺-tetracycline interfered with cleavage by Fe²⁺. These findings suggest that cleavage results from binding at a substrate-specific site. Fe²⁺ is known to be able to cleave amide bonds in proteins at distances up to approximately 12 Å. We conclude that the carbon of glutamine 225 is probably within 12 Å of the position of the Fe²⁺ ion in the Fe²⁺-tetracycline complex bound to the protein.

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* Corresponding author. Mailing address: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-6764. Fax: (617) 636-0458. E-mail: laura.mcmurry{at}tufts.edu.

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Journal of Bacteriology, September 2002, p. 5113-5120, Vol. 184, No. 18
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.18.5113-5120.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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