Tetracycline-chelated Mg2+ ion initiates helix unwinding in tet repressor induction

被引:57
作者
Orth, P [1 ]
Saenger, W [1 ]
Hinrichs, W [1 ]
机构
[1] Free Univ Berlin, Inst Kristallog, D-14195 Berlin, Germany
关键词
D O I
10.1021/bi9816610
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 Angstrom 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 N epsilon 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 O gamma of Thr103. The induced 2.5 Angstrom movement of Thr103 results in the partial unwinding of helix alpha(6), associated with a lateral shift of helices alpha(4) and alpha(9). 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.
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页码:191 / 198
页数:8
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