Instituto de Química-Física Rocasolano, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.
Nucleic Acids Res. 2010 Aug;38(15):5226-41. doi: 10.1093/nar/gkq277. Epub 2010 Apr 21.
Direct targeting of critical DNA-binding elements of a repressor by its cognate antirepressor is an effective means to sequester the repressor and remove a transcription initiation block. Structural descriptions for this, though often proposed for bacterial and phage repressor-antirepressor systems, are unavailable. Here, we describe the structural and functional basis of how the Myxococcus xanthus CarS antirepressor recognizes and neutralizes its cognate repressors to turn on a photo-inducible promoter. CarA and CarH repress the carB operon in the dark. CarS, produced in the light, physically interacts with the MerR-type winged-helix DNA-binding domain of these repressors leading to activation of carB. The NMR structure of CarS1, a functional CarS variant, reveals a five-stranded, antiparallel beta-sheet fold resembling SH3 domains, protein-protein interaction modules prevalent in eukaryotes but rare in prokaryotes. NMR studies and analysis of site-directed mutants in vivo and in vitro unveil a solvent-exposed hydrophobic pocket lined by acidic residues in CarS, where the CarA DNA recognition helix docks with high affinity in an atypical ligand-recognition mode for SH3 domains. Our findings uncover an unprecedented use of the SH3 domain-like fold for protein-protein recognition whereby an antirepressor mimics operator DNA in sequestering the repressor DNA recognition helix to activate transcription.
直接靶向抑制剂的关键 DNA 结合元件是一种有效的方法来隔离抑制剂并去除转录起始障碍。尽管经常为细菌和噬菌体抑制剂 - 抗抑制剂系统提出结构描述，但目前还没有。在这里，我们描述了粘细菌 CarS 抗抑制剂如何识别和中和其同源抑制剂以开启光诱导启动子的结构和功能基础。在黑暗中，CarA 和 CarH 抑制 carB 操纵子。在光下产生的 CarS 与这些抑制剂的 MerR 型翼螺旋 DNA 结合域发生物理相互作用，导致 carB 的激活。CarS1 的 NMR 结构，一种功能性的 CarS 变体，揭示了一个五链，反平行的β-折叠结构类似于 SH3 结构域，这是真核生物中普遍存在但在原核生物中罕见的蛋白质 - 蛋白质相互作用模块。NMR 研究和体内及体外的定点突变分析揭示了 CarS 中一个暴露在溶剂中的疏水性口袋，其中含有酸性残基，CarA 的 DNA 识别螺旋以非典型的配体识别模式与 SH3 结构域高亲和力对接。我们的发现揭示了一种前所未有的使用 SH3 结构域样折叠进行蛋白质 - 蛋白质识别的方法，其中抗抑制剂模拟操作员 DNA 以隔离抑制剂 DNA 识别螺旋以激活转录。
