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首页> 外文期刊>Journal of Molecular Biology >The 3D Solution Structure of the C-terminal Region of Ku86 (Ku86CTR).
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The 3D Solution Structure of the C-terminal Region of Ku86 (Ku86CTR).

机译：Ku86（Ku86CTR）C端区域的3D解决方案结构。

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In eukaryotes the non-homologous end-joining repair of double strand breaks in DNA is executed by a series of proteins that bring about the synapsis, preparation and ligation of the broken DNA ends. The mechanism of this process appears to be initiated by the obligate heterodimer (Ku70/Ku86) protein complex Ku that has affinity for DNA ends. Ku then recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The three-dimensional structures of the major part of the Ku heterodimer, representing the DNA-binding core, both free and bound to DNA are known from X-ray crystallography. However, these structures lack a region of ca 190 residues from the C-terminal region (CTR) of the Ku86 subunit (also known as Lupus Ku autoantigen p86, Ku80, or XRCC5) that includes the extreme C-terminal tail that is reported to be sufficient for DNA-PKcs-binding. We have examined the structural characteristics of the Ku86CTR protein expressed in bacteria. By deletion mutagenesis and heteronuclear NMR spectroscopywe localised a globular domain consisting of residues 592-709. Constructs comprising additional residues either to the N-terminal side (residues 543-709), or the C-terminal side (residues 592-732), which includes the putative DNA-PKcs-binding motif, yielded NMR spectra consistent with these extra regions lacking ordered structure. The three-dimensional solution structure of the core globular domain of the C-terminal region of Ku86 (Ku86CTR(592-709)) has been determined using heteronuclear NMR spectroscopy and dynamical simulated annealing using structural restraints from nuclear Overhauser effect spectroscopy, and scalar and residual dipolar couplings. The polypeptide fold comprises six regions of alpha-helical secondary structure that has an overall superhelical topology remotely homologous to the MIF4G homology domain of the human nuclear cap binding protein 80 kDa subunit and the VHS domain of the Drosophila protein Hrs, though strict analysis of the structures suggests that these domains are not functionally related. Two prominent hydrophobic pockets in the gap between helices alpha2 and alpha4 suggest a potential ligand-binding characteristic for this globular domain.
机译：在真核生物中，DNA中双链断裂的非同源末端连接修复是通过一系列蛋白质实现的，这些蛋白质引起突触，DNA断裂的制备和连接。此过程的机制似乎是由对DNA末端具有亲和力的专性异二聚体（Ku70 / Ku86）蛋白复合物Ku引发的。然后，Ku募集了DNA依赖性蛋白激酶催化亚基（DNA-PKcs）。从X射线晶体学已知Ku异二聚体的主要部分的三维结构，其代表DNA结合核心，既游离又结合到DNA。但是，这些结构缺少距Ku86亚基（也称为狼疮Ku自身抗原p86，Ku80或XRCC5）的C端区域（CTR）大约190个残基的区域，该区域据报道包括足以进行DNA-PKcs结合。我们已经检查了在细菌中表达的Ku86CTR蛋白的结构特征。通过缺失诱变和异核NMR光谱我们定位了一个由残基592-709组成的球形结构域。在N端（残基543-709）或C端（残基592-732）包含附加残基的构建体，其包含推定的DNA-PKcs结合基序，产生与这些额外区域一致的NMR光谱缺乏有序的结构。 Ku86（Ku86CTR（592-709））的C末端区域的核心球状结构域的三维溶液结构已使用异核NMR光谱和动态模拟退火方法进行了测定，所述动力学模拟退火使用了核Overhauser效应光谱技术以及标量和残留的偶极耦合。多肽折叠包含六个α-螺旋二级结构区域，该区域具有与人核帽结合蛋白80 kDa亚基的MIF4G同源结构域和果蝇蛋白Hrs的VHS结构域远程同源的总体超螺旋拓扑结构，结构表明这些域在功能上不相关。螺旋α2和α4之间的间隙中有两个突出的疏水口袋，表明该球状结构域具有潜在的配体结合特性。

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