title: Structural origins for selectivity and specificity in an engineered bacterial repressor-inducer pair.
abstract: The bacterial tetracycline transcription regulation system mediated by the tetracycline repressor (TetR) is widely used to study gene expression in prokaryotes and eukaryotes. To study multiple genes in parallel, a triple mutant TetR(K(64)L(135)I(138)) has been engineered that is selectively induced by the synthetic tetracycline derivative 4-de-dimethylamino-anhydrotetracycline (4-ddma-atc) and no longer by tetracycline, the inducer of wild-type TetR. In the present study, we report the crystal structure of TetR(K(64)L(135)I(138)) in the absence and in complex with 4-ddma-atc at resolutions of 2.1 A. Analysis of the structures in light of the available binding data and previously reported TetR complexes allows for a dissection of the origins of selectivity and specificity. In all crystal structures solved to date, the ligand-binding position, as well as the positioning of the residues lining the binding site, is extremely well conserved, irrespective of the chemical nature of the ligand. Selective recognition of 4-ddma-atc is achieved through fine-tuned hydrogen-bonding constraints introduced by the His64-->Lys substitution, as well as a combination of hydrophobic effect and the removal of unfavorable electrostatic interactions through the introduction of Leu135 and Ile138.
authors.affiliation: Department of Biology, Lehrstuhl für Biotechnik, Friedrich-Alexander University, Erlangen-Nuremberg, Germany.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Klieber
authors.forename: Oliver
authors.initials: O
authors.lastname: Scholz
authors.forename: Susanne
authors.initials: S
authors.lastname: Lochner
authors.forename: Peter
authors.initials: P
authors.lastname: Gmeiner
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hillen
authors.forename: Yves A
authors.initials: YA
authors.lastname: Muller
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055672:Static Electricity; D013752:Tetracycline / Q000494:pharmacology; D013753:Tetracycline Resistance
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; D011994:Recombinant Proteins; D012097:Repressor Proteins; C053450:4-des-dimethylaminotetracycline; D013752:Tetracycline