Abstract

We constructed a mutant of the tetracycline-inducible repressor protein TetR with specificity for the tc analogue 4-de(dimethylamino)anhydrotetracycline (4-ddma-atc), which is neither an antibiotic nor an inducer for the wild-type protein. The previously published relaxed specificity mutant TetR H64K S135L displays reduced induction by tc but full induction by doxycycline (dox), anhydrotetracycline (atc), and 4-de(dimethylamino)-6-demethyl-6-deoxytetracycline (cmt3). To create induction specificity for tc derivatives lacking the 4-dimethylamino grouping such as cmt3 and 4-ddma-atc, the residues at positions 82 and 138, which are located close to that moiety in the crystal structure of the TetR-[tc-Mg](+)(2) complex, were randomized. We anticipated that a residue with increased size may lead to sterical hindrance, and screening for 4-ddma-atc-specific induction indeed revealed the mutant TetR H64K S135L S138I. Out of 24 exchanges only the addition of S138I to TetR H64K S135L yielded a mutant with a pronounced reduction of affinity for atc and dox, while the one for 4-ddma-atc is not affected. The ratio of binding constants revealed a 200-fold specificity increase for 4-ddma-atc over atc. The contributions of each single mutant to specificity indicate that the tc variants bind slightly different positions in the TetR tc binding pocket.