Fe2+-Tetracycline-Mediated Cleavage of the Tn10Tetracycline Efflux Protein TetA Reveals a Substrate Binding Site near Glutamine 225 in Transmembrane Helix 7

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Abstract
TetA specified by Tn10is 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 Mg2+-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 α 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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