Dunham Thomas D, Xu Weijun, Funnell Barbara E, Schumacher Maria A
Department of Biochemistry and Molecular Biology, MD Anderson Cancer Center, University of Texas, Houston, TX 77030, USA.
EMBO J. 2009 Jun 17;28(12):1792-802. doi: 10.1038/emboj.2009.120. Epub 2009 May 21.
The accurate segregation of DNA is essential for the faithful inheritance of genetic information. Segregation of the prototypical P1 plasmid par system requires two proteins, ParA and ParB, and a centromere. When bound to ATP, ParA mediates segregation by interacting with centromere-bound ParB, but when bound to ADP, ParA fulfils a different function: DNA-binding transcription autoregulation. The structure of ParA is unknown as is how distinct nucleotides arbitrate its different functions. To address these questions, we carried out structural and biochemical studies. Crystal structures show that ParA consists of an elongated N-terminal alpha-helix, which unexpectedly mediates dimerization, a winged-HTH and a Walker-box containing C-domain. Biochemical data confirm that apoParA forms dimers at physiological concentrations. Comparisons of four apoParA structures reveal a strikingly flexible dimer interface that allows ParA to adopt multiple conformations. The ParA-ADP structure shows that ADP-binding activates DNA binding using a bipartite mechanism. First, it locks in one specific dimer conformation, and second, it induces the folding of two DNA-binding basic motifs that we show are critical for operator binding.
DNA的准确分离对于遗传信息的忠实遗传至关重要。典型的P1质粒par系统的分离需要两种蛋白质，即ParA和ParB，以及一个着丝粒。当与ATP结合时，ParA通过与着丝粒结合的ParB相互作用介导分离，但当与ADP结合时，ParA发挥不同的功能：DNA结合转录自调控。ParA的结构未知，不同的核苷酸如何决定其不同功能也不清楚。为了解决这些问题，我们进行了结构和生化研究。晶体结构表明，ParA由一个细长的N端α螺旋、一个翼状HTH和一个包含C结构域的沃克盒组成，其中N端α螺旋意外地介导二聚化。生化数据证实，脱辅基ParA在生理浓度下形成二聚体。对四种脱辅基ParA结构的比较揭示了一个极其灵活的二聚体界面，使ParA能够采用多种构象。ParA-ADP结构表明，ADP结合通过一种双重机制激活DNA结合。首先，它锁定在一种特定的二聚体构象中，其次，它诱导两个DNA结合碱性基序的折叠，我们发现这两个基序对于操纵子结合至关重要。
