THE COMPLEX FORMED BETWEEN TET REPRESSOR AND TETRACYCLINE-MG2-RESISTANCE( REVEALS MECHANISM OF ANTIBIOTIC)

In recent years Gram-negative bacteria have developed several resistan ce mechanisms against the broad-spectrum antibiotic tetracycline (Tc). The most abundant mechanism involves a membrane-associated protein (T etA) that exports the antibiotic out of the bacterial cell before it c an attach to the ribosomes and inhibit polypeptide elongation. The exp ression of the TetA protein is regulated by the Tet repressor (TetR). It occurs as a homodimer and binds with two alpha-helix-turn-alpha-hel ix motifs (HTH) to two tandemly orientated DNA operators, thereby bloc king 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 unab le to bind to DNA. TetR of class D, TetR(D), was cocrystallized with t etracycline (7HTc) and Mg2+ in space group I4(1)22 and studied by X-ra y diffraction. One TetR(D) monomer occupies the crystal asymmetric uni t, and the dimer is formed by a crystallographic 2-fold rotation. The crystal structure was determined by multiple isomorphous replacement a t 2.5 Angstrom resolution, and on this basis the structure of the near ly isomorphous complex with 7-chlorotetracycline, TetR(D)/(Mg 7ClTc)(), has been refined to an X-factor of 18.3 % using all reflections to 2.1 Angstrom resolution. TetR(D) folds into ten alpha-helices with con necting turns and loops. The N-terminal three alpha-helices of the rep ressor form the DNA-binding domain, including the HTH with an inverse orientation compared with HTH in other DNA-binding proteins. The dista nce of 39 Angstrom between the two recognition helices explains the in ability of the induced TetR to bind to B-form DNA. The core of the pro tein is formed by helices alpha 5 to alpha 10. It is responsible for d imerization and contains, for each monomer, a binding pocket that acco mmodates Tc in the presence of a divalent cation. The structure of the TetR(D)/(Mg 7ClTc)(+) complex reveals the octahedral coordination of Mg2+ by Tc (chelating O-11, and O-12), His100 N-e and by three water m olecules; in addition there is an extended network of hydrogen bonding and van der Waals interactions formed between 7ClTe and TetR. The det ailed view of the Tc-binding pocket and the interactions between the a ntibiotic and the repressor offers the first solid basis for rational tetracycline design, with the aim of circumventing resistance.