Henssler Eva-Maria, Scholz Oliver, Lochner Susanne, Gmeiner Peter, Hillen Wolfgang
Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany.
Biochemistry. 2004 Jul 27;43(29):9512-8. doi: 10.1021/bi049682j.
We constructed a mutant of the tetracycline-inducible repressor protein TetR with specificity for the tc analogue 4-de(dimethylamino)anhydrotetracycline (4-ddma-atc), which is neither an antibiotic nor an inducer for the wild-type protein. The previously published relaxed specificity mutant TetR H64K S135L displays reduced induction by tc but full induction by doxycycline (dox), anhydrotetracycline (atc), and 4-de(dimethylamino)-6-demethyl-6-deoxytetracycline (cmt3). To create induction specificity for tc derivatives lacking the 4-dimethylamino grouping such as cmt3 and 4-ddma-atc, the residues at positions 82 and 138, which are located close to that moiety in the crystal structure of the TetR-tc-Mg(2) complex, were randomized. We anticipated that a residue with increased size may lead to sterical hindrance, and screening for 4-ddma-atc-specific induction indeed revealed the mutant TetR H64K S135L S138I. Out of 24 exchanges only the addition of S138I to TetR H64K S135L yielded a mutant with a pronounced reduction of affinity for atc and dox, while the one for 4-ddma-atc is not affected. The ratio of binding constants revealed a 200-fold specificity increase for 4-ddma-atc over atc. The contributions of each single mutant to specificity indicate that the tc variants bind slightly different positions in the TetR tc binding pocket.
我们构建了一种对四环素类似物4-去（二甲基氨基）脱水四环素（4-ddma-atc）具有特异性的四环素诱导型阻遏蛋白TetR突变体，4-ddma-atc既不是抗生素，也不是野生型蛋白的诱导剂。先前发表的宽松特异性突变体TetR H64K S135L对四环素的诱导作用降低，但对强力霉素（dox）、脱水四环素（atc）和4-去（二甲基氨基）-6-去甲基-6-脱氧四环素（cmt3）有完全诱导作用。为了对缺乏4-二甲基氨基基团的四环素衍生物（如cmt3和4-ddma-atc）产生诱导特异性，对TetR-tc-Mg(2)复合物晶体结构中靠近该部分的82位和138位残基进行了随机化。我们预计，尺寸增加的残基可能会导致空间位阻，对4-ddma-atc特异性诱导的筛选确实揭示了突变体TetR H64K S135L S138I。在24种交换中，只有在TetR H64K S135L中添加S138I才能产生对atc和dox亲和力显著降低的突变体，而对4-ddma-atc的亲和力不受影响。结合常数的比值显示，4-ddma-atc对atc的特异性增加了200倍。每个单一突变体对特异性的贡献表明，四环素变体在TetR四环素结合口袋中的结合位置略有不同。
