Tetracycline Analogs Affecting Binding to Tn10-Encoded Tet Repressor Trigger the Same Mechanism of Induction

Abstract

We examined the influence of substituents in tetracycline (tc) analogs modified at positions 2 and 4-9 and anhydrotetracycline (atc) on induction of the Tn10-encoded Tet repressor (TetR) by a quantitative in vitro induction assay. The equilibrium association constants of the modified tc to TetR were independently determined to distinguish effects on binding from those on induction. We found a correlation between the binding affinity and induction of TetR for most tc analogs. While a substitution at position 5 revealed only minor effects, changes at position 6 increased binding and induction efficiencies up to 20-fold. A chlorine at position 7 or 8 enhanced binding and induction about 4- and 9-fold, respectively. Substituents at position 9 decreased binding up to 5-fold. Epimerization of the dimethylamino function at position 4 in 4-epi-tc resulted in about 300-fold-reduced binding and 80-fold-reduced induction. Substitution of this grouping by hydrogen in 4-de(dimethylamino)-tc resulted in no binding and no induction. The respective atc analog failed to induce as well, although binding was still observed. The dimethylamino function may, thus, play a role in triggering the conformational change of TetR necessary for induction. Substitution of the 2-carboxamido by a nitrilo function did not influence binding and induction efficiencies. Atc showed about 30-fold increased binding and induction, being the most effective inducer tested in this study. The equilibrium association constants of most TetR-[Mg-tc]+ and TetR-([Mg-tc]+)2 analog complexes with tet operator are decreased about 10(2)- and 10(8)-fold, respectively, as compared to those of free TetR. This suggests that these tc analogs share the same molecular mechanism of TetR induction.

The Role of the Variable Region in Tet Repressor for Inducibility by Tetracycline

“…Substitutions at positions 7, 8, or 9 of tc that contact amino acids in ␣9 affect TetR binding and induction less than 10-fold (35,39). Twelve induction-deficient TetR mutants were isolated in ␣9.…”

“…Free Mg 2ϩ concentrations ranging from 10 Ϫ7 to 10 Ϫ2 M were adjusted using diluted stock solutions of MgCl 2 and O buffer (100 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 5 mM dithiothreitol) without EDTA. A K M of 2400 M Ϫ1 for the association of Mg 2ϩ to tc was employed to analyze the titration curves (35). The fitting was performed by minimizing S 2 ϭ ⌺(F exp Ϫ F theor ) 2 , where F exp and F theor are the experimental and theoretical fluorescence intensities, respectively.…”

Molecular mechanics models for tetracycline analogs

“…Indeed, it was found experimentally that 4-ddmaTc does not bind to TetR of class B. 36 It probably does not bind to TetRs of other classes, because His64, Gln116, and Asn82 are strictly conserved among all TetR classes. 8 …”

Delay in resumption of the activity of tetracycline-regulatable promoter following removal of tetracycline analogues

“…For most tetracycline analogues, this efficiency has been shown to in vivo applications, a more rapid resumption of tRP activity may be achieved by using oxytetracycline rather be correlated with the association equilibrium constant (K A ) of the analogue for tetR. 25 , 26 respectively. K A for minocycline is lower than that of 4-epi-7-chloro-tetracycof doxycycline is clearly more convenient.…”