Orth P, Saenger W, Hinrichs W
Institut für Kristallographie, Freie Universität Berlin, Germany.
Biochemistry. 1999 Jan 5;38(1):191-8. doi: 10.1021/bi9816610.
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.
同二聚体四环素阻遏蛋白（TetR）在转录水平上调节对抗生素四环素的抗性。在没有四环素（Tc）的情况下，TetR利用两个螺旋-转角-螺旋（HTH）基序与回文操纵序列结合。如果四环素-Mg2+复合物[MgTc]+进入埋藏在TetR同二聚体内的两个相同的结合通道，就会发生构象变化，诱导形成的[TetR/[MgTc]+]2复合物会释放操纵子DNA。为了证明Mg2+对[MgTc]+结合和TetR诱导的作用，通过添加乙二胺四乙酸（EDTA）逐步降低诱导的TetR同二聚体中的Mg2+浓度，得到了无Mg2+和半占据的TetR（D）的两个X射线晶体结构。尽管完全或部分没有Mg2+，Tc仍与[MgTc]+结合位点结合。将这些结构与无诱导剂的TetR（D）一起精修至2.2至2.7埃的分辨率，并与与两个[MgTc]+形成复合物的完全诱导的TetR（D）进行比较。每个诱导剂结合通道有三个组成部分，一个疏水部分和两个亲水部分。其中一个亲水接触区域通过氢键结合Tc；疏水区域正确定位Tc并部分封闭结合通道的入口；第二个亲水区域配位Mg2+，传导诱导信号，并完成封闭通道入口的过程。Tc赋予TetR结合特异性，而Mg2+主要负责诱导：Mg2+与His100的咪唑Nε结合后，以八面体形式与Tc的1,3-酮烯醇基团和三个水分子配位。其中一个水分子与Thr103的羟基Oγ形成氢键。Thr103诱导的2.5埃移动导致α6螺旋部分解旋，与α4和α9螺旋的横向移动相关。它们同时封闭通道入口，并使DNA结合结构域采取非结合构象，导致操纵子DNA释放以及负责抗性的基因表达。
