The Complex Formed Between Tet Repressor and Tetracycline-Mg2|ihsbop|+Reveals Mechanism of Antibiotic Resistance

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Abstract

In recent years Gram-negative bacteria have developed several resistance mechanisms against the broad-spectrum antibiotic tetracycline (Tc). The most abundant mechanism involves a membrane-associated protein (TetA) that exports the antibiotic out of the bacterial cell before it can attach to the ribosomes and inhibit polypeptide elongation. The expression of the TetA protein is regulated by the Tet repressor (TetR). It occurs as a homodimer and binds with two α-helix-turn-α-helix motifs (HTH) to two tandemly orientated DNA operators, thereby blocking the expression of the associated genes, one encoding for TetA and the other for TetR. If Tc in complex with a divalent cation binds to TetR, a conformational change occurs and the induced TetR is then unable to bind to DNA. TetR of class D, TetR|super|D, was cocrystallized with tetracycline (7HTc) and Mg2|ihsbop|+in space groupI4|infer|1|2ru|22 and studied by X-ray diffraction. One TetR|super|D monomer occupies the crystal asymmetric unit, and the dimer is formed by a crystallographic 2-fold rotation. The crystal structure was determined by multiple isomorphous replacement at 2.5|4ru|Å resolution, and on this basis the structure of the nearly isomorphus complex with 7-chlorotetracycline, TetR|super|D/(Mg 7CITc)|superihsbop|+, has been refined to anR-factor of 18.2% using all reflections to 2.1|4ru|Å resolution. TetR|super|D folds into ten α-helices with connecting turns and loops. The N-terminal three α-helices of the repressor form the DNA-binding domain, including the HTH with an inverse orientation compared with HTH in other DNA-binding proteins. The distance of 39|4ru|Å between the two recognition helices explains the inability of the induced TetR to bind toB-form DNA. The core of the protein is formed by helices α5 to α10. It is responsible for dimerization and contains, for each monomer, a binding pocket that accommodates Tc in the presence of a divalent cation The structure of the TetT|super|D/(Mg 7CITc)|superihsbop|+ complex reveals the octahedral coordination of Mg|superopnbop|2|ihsbop|+|clobop| by Tc (chelating O-11, and O-12), His100 N|superlcepsil| and by three water molecules; in addition there is an extended network of hydrogen bonding and van der Waals intractions formed between 7CITc and TetR. The detailed view of the Tc-binding pocket and the interactions between the antibiotic and the repressor offers the first solid basis for rational tetracycline design, with the aim of circumventing resistance.

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