Teaching TetR to recognize a new inducer

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Authors

  • Oliver Scholz
  • Martin Köstner
  • Marco Reich
  • Susanne Gastiger
  • Wolfgang Hillen

Tet Repressor (TetR) recognizes the inducer tetracycline (tc) with high affinity. The tc analog 4-de(dimethylamino)-6-deoxy-6-demethyl-tetracycline (cmt3) is not an inducer for TetR. Induction specificity for cmt3 was generated by employing a directed evolution approach to screen appropriate TetR mutants in four successive steps. The specificity of the best TetR mutant is more than 20,000-fold increased for cmt3 over tc as judged by the ratio of their respective binding constants. Two rounds of directed evolution via DNA shuffling revealed His64 as a key residue for inducer specificity. The best TetR mutant with cmt3 specificity contains the H64K exchange, leading to a 300-fold decreased tc and a 20-fold increased cmt3 affinity. Another round of directed evolution made use of randomized oligonucleotides to mutate selected residues close to the tc-binding pocket of TetR and yielded TetR S135L with a 250-fold increased cmt3 affinity. The double mutant TetR H64K S135L was constructed and again subjected to directed evolution using randomized oligonucleotides to alter residues in the "secondary shell" of the tc-binding pocket. The resulting best mutants TetR H64K E114Q S135L, TetR A61V H64K Q109E Q116E S135L and TetR H64K T112K S135L are fully inducible by cmt3 and not by tc. Thus, their inducer specificity has been redesigned. The molecular mechanism of changed inducer recognition is discussed, based on binding constants with several tc analogs and in light of the TetR crystal structure.

Original languageEnglish
JournalJournal of Molecular Biology
Volume329
Issue number2
Pages (from-to)217-227
Number of pages11
ISSN0022-2836
DOIs
Publication statusPublished - 30.05.2003
Externally publishedYes

Bibliographical note

We thank Brad Zerler, CollaGenex, PA, for providing cmt1 and cmt3, and Mark Nelson, Paratek, Boston, MA, for san. This work was supported by the Deutsche Forschungsgemeinschaft through the SFB 473 and the Fonds der Chemischen Industrie. O.S. obtained a personal grant from the the Fonds der Chemischen Industrie.

    Research areas

  • cmt3, Inducer specificity, Induction, Tet repressor, Tetracycline
  • Chemistry