Structural studies of the high mobility group globular domain and basic tail of HMG-D bound to disulfide cross-linked DNA
Structural studies of the high mobility group globular domain and basic tail of HMG-D bound to disulfide cross-linked DNA
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DOI：
10.1021/bi000723v
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发表时间：
2000-08-15
期刊：
影响因子：
2.9
通讯作者：
Churchill, MEA
中科院分区：
文献类型：
--
作者：
Dow, LK;Jones, DNM;Churchill, MEA
HMG-D是一种存在于果蝇早期胚胎发育过程中的高迁移率族染色体蛋白，是一个非序列特异性的蛋白家族成员，它利用HMG结构域与小沟中的DNA结合。HMG-D的高电荷C末端尾部含有AK基序，其有助于高亲和力非序列特异性DNA结合。为了理解HMG结构域和HMG-D的C-末端尾部与溶液中DNA的相互作用，使用异源NMR技术研究了高亲和力截短形式的蛋白质和二硫键交联的DNA片段之间的复合物。尽管HMG-D对DNA上单个“预弯曲”位点的亲和力相对较高（Kd = 1.4 nM），但HMG-D与DNA形成非序列特异性复合物，如溶液中DNA界面处蛋白质共振的交换加宽所示。当蛋白质与DNA复合时，蛋白质的二级结构元件被保留，并且DNA结合界面映射到发生最大化学位移差异的蛋白质区域。HMG-D的C-末端尾部赋予高亲和力DNA结合，具有未定义的结构，并且似乎通过可能受磷酸化调节的残基在DNA的大沟中直接接触。我们的结论是，虽然HMG-D的HMG结构域识别DNA的结合模式类似于序列特异性HMG结构域转录因子所使用的模式，但DNA结合结构域的C-末端和C-末端尾部的结构和相互作用存在显著差异。
HMG-D is an abundant high mobility group chromosomal protein present during early embryogenesis in Drosophila melanogaster, It is a non-sequence-specific member of a protein family that uses the HMG domain for binding to DNA in the minor groove. The highly charged C-terminal tail of HMG-D contains AK motifs that contribute to high-affinity non-sequence-specific DNA binding. To understand the interactions of the HMG domain and C-terminal tail of HMG-D with DNA in solution, a complex between a high-affinity truncated form of the protein and a disulfide cross-linked DNA fragment was studied using heteronuclear NMR techniques. Despite its relatively high affinity for the single "prebent" site on the DNA, K-d = 1.4 nM, HMG-D forms a non-sequence-specific complex with the DNA as indicated by exchange broadening of the protein resonances at the DNA interface in solution. The secondary structural elements of the protein are preserved when the protein is complexed with the DNA, and the DNA-binding interface maps to the regions of the protein where the largest chemical shift differences occur. The C-terminal tail of HMG-D confers high-affinity DNA binding, has an undefined structure, and appears to make direct contacts in the major groove of DNA via residues that are potentially regulated by phosphorylation. We conclude that while the HMG domain of HMG-D recognizes DNA with a mode of binding similar to that used by the sequence-specific HMG domain transcription factors, there are noteworthy differences in the structure and interactions of the C-terminal end of the DNA-binding domain and the C-terminal tail.