title: Tetracycline-chelated Mg2+ ion initiates helix unwinding in Tet repressor induction.
abstract: The homodimeric tetracycline repressor (TetR) regulates resistance to the antibiotic tetracycline at the transcriptional level. TetR binds in the absence of Tc to palindromic operator sequences utilizing two helix-turn-helix (HTH) motifs. If the tetracycline-Mg2+ complex [MgTc]+ enters two identical binding tunnels buried within the TetR homodimer, a conformational change takes place, and the induced [TetR/[MgTc]+]2 complex releases operator DNA. To demonstrate the contribution of Mg2+ to [MgTc]+ binding and TetR induction, the Mg2+ concentration in the induced TetR homodimer was progressively reduced by addition of EDTA, resulting in two X-ray crystal structures of Mg2+-free and half-occupied TetR(D). Tc remains bound to the [MgTc]+-binding sites, despite the complete or partial absence of Mg2+. Together with inducer-free TetR(D), the structures were refined to between 2.2 and 2.7 A resolution and compared with fully induced TetR(D) in complex with two [MgTc]+. Each inducer binding tunnel has three constituent parts, one hydrophobic and two hydrophilic ones. One of the hydrophilic contact areas binds Tc by hydrogen bonding; the hydrophobic region correctly positions Tc and partially closes the entrance to the binding tunnel; the second hydrophilic region coordinates Mg2+, transduces the induction signal, and completes the process of closing the tunnel entrance. Tc confers binding specificity to TetR while Mg2+ is primarily responsible for induction: After binding to the imidazole Nepsilon of His100, Mg2+ is octahedrally coordinated to the 1,3-ketoenolate group of Tc and to three water molecules. One of these waters forms a hydrogen bond to the hydroxyl group Ogamma of Thr103. The induced 2.5 A movement of Thr103 results in the partial unwinding of helix alpha6, associated with a lateral shift of helices alpha4 and alpha9. They simultaneously close the tunnel entrance and cause the DNA-binding domains to adopt a nonbinding conformation, leading to release of operator DNA and expression of the genes responsible for resistance.
authors.affiliation: Institut für Kristallographie, Freie Universität Berlin, Germany.
authors.forename: P
authors.initials: P
authors.lastname: Orth
authors.forename: W
authors.initials: W
authors.lastname: Saenger
authors.forename: W
authors.initials: W
authors.lastname: Hinrichs
mesh_terms: D002614:Chelating Agents / Q000737:chemistry*; D003198:Computer Simulation; D003460:Crystallization; D018360:Crystallography, X-Ray; D019281:Dimerization; D019077:Helix-Turn-Helix Motifs; D046911:Macromolecular Substances; D008274:Magnesium / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D013752:Tetracycline / Q000737:chemistry*
chemical_list: D002614:Chelating Agents; D046911:Macromolecular Substances; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D013752:Tetracycline; D008274:Magnesium