Kisker C, Hinrichs W, Tovar K, Hillen W, Saenger W
Institut für Kristallographie, Freie Universität Berlin, Germany.
J Mol Biol. 1995 Mar 24;247(2):260-80. doi: 10.1006/jmbi.1994.0138.
In recent years Gram-negative bacteria have developed several resistance mechanisms against the broad-spectrum antibiotic tetracycline (Tc). The most abundant mechanism involves a membrane-associated protein (TetA) that exports the antibiotic out of the bacterial cell before it can attach to the ribosomes and inhibit polypeptide elongation. The expression of the TetA protein is regulated by the Tet repressor (TetR). It occurs as a homodimer and binds with two alpha-helix-turn-alpha-helix motifs (HTH) to two tandemly orientated DNA operators, thereby blocking the expression of the associated genes, one encoding for TetA and the other for TetR. If Tc in complex with a divalent cation binds to TetR, a conformational change occurs and the induced TetR is then unable to bind to DNA. TetR of class D, TEtRD, was cocrystallized with tetracycline (7HTc) and Mg2+ in space group I4(1)22 and studied by X-ray diffraction. One TetRD monomer occupies the crystal asymmetric unit, and the dimer is formed by a crystallographic 2-fold rotation. The crystal structure was determined by multiple isomorphous replacement at 2.5 A resolution, and on this basis the structure of the nearly isomorphous complex with 7-chlorotetracycline, TetRD/(Mg 7CITc)+, has been refined to an R-factor of 18.3% using all reflections to 2.1 A resolution. TetRD folds into ten alpha-helices with connecting turns and loops. The N-terminal three alpha-helices of the repressor form the DNA-binding domain, including the HTH with an inverse orientation compared with HTH in other DNA-binding proteins. The distance of 39 A between the two recognition helices explains the inability of the induced TetR to bind to B-form DNA. The core of the protein is formed by helices alpha 5 to alpha 10. It is responsible for dimerization and contains, for each monomer, a binding pocket that accommodates Tc in the presence of a divalent cation. The structure of the TetRD/(Mg 7CITc)+ complex reveals the octahedral coordination of Mg2+ by Tc (chelating O-11, and O-12), His100 N epsilon and by three water molecules; in addition there is an extended network of hydrogen bonding and van der Waals interactions formed between 7CITc and TetR. The detailed view of the Tc-binding pocket and the interactions between the antibiotic and the repressor offers the first solid basis for rational tetracycline design, with the aim of circumventing resistance.
近年来，革兰氏阴性菌已形成了几种针对广谱抗生素四环素（Tc）的耐药机制。最常见的机制涉及一种与膜相关的蛋白质（TetA），它能在抗生素附着到核糖体并抑制多肽延伸之前将其输出细菌细胞。TetA蛋白的表达受Tet阻遏物（TetR）调控。TetR以同二聚体形式存在，并通过两个α-螺旋-转角-α-螺旋基序（HTH）与两个串联排列的DNA操纵子结合，从而阻断相关基因的表达，一个基因编码TetA，另一个编码TetR。如果与二价阳离子结合的Tc与TetR结合，就会发生构象变化，诱导的TetR随后无法与DNA结合。D类TetR（TEtRD）与四环素（7HTc）和Mg2+在空间群I4(1)22中进行了共结晶，并通过X射线衍射进行研究。一个TetRD单体占据晶体不对称单元，二聚体通过晶体学2次旋转形成。晶体结构通过多重同晶置换在2.5 Å分辨率下确定，在此基础上，与7-氯四环素形成的近乎同晶的复合物TetRD/(Mg 7CITc)+，利用所有2.1 Å分辨率的反射将其精修至R因子为18.3%。TetRD折叠成十个带有连接转角和环的α-螺旋。阻遏物N端的三个α-螺旋形成DNA结合结构域，其中包括与其他DNA结合蛋白中的HTH方向相反的HTH。两个识别螺旋之间39 Å的距离解释了诱导的TetR无法与B型DNA结合的原因。蛋白质的核心由α5至α10螺旋形成。它负责二聚化，并且对于每个单体而言，都包含一个在存在二价阳离子时容纳Tc的结合口袋。TetRD/(Mg 7CITc)+复合物的结构揭示了Tc（螯合O-11和O-12）、His100 Nε以及三个水分子对Mg2+的八面体配位；此外，7CITc与TetR之间形成了广泛的氢键和范德华相互作用网络。Tc结合口袋的详细视图以及抗生素与阻遏物之间的相互作用为合理设计四环素提供了首个坚实基础，目的是规避耐药性。
