Structural analysis of the DNA target site and its interaction with Mbp1
Structural analysis of the DNA target site and its interaction with Mbp1
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DOI：
10.1039/b912309a
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发表时间：
2009-01-01
影响因子：
3.2
通讯作者：
Lane, Andrew N.
中科院分区：
文献类型：
--
作者：
Chernatynskaya, Anna V.;Deleeuw, Lynn;Lane, Andrew N.
含有酵母转录因子 Mbp1 共有结合位点的 14 碱基对非自互补 DNA 双链体的溶液结构已通过 NMR 确定，该双链体使用标量耦合分析、时间依赖性 NOE、残余偶极耦合和用 C-13 核苷酸均匀标记的一条链制备的双链体的 C-13 编辑 NMR 光谱。正如预期的那样，游离 DNA 双链体属于 B 族结构，并且在实验限制内是直的。然而，结合序列中存在与共有 CGCG 元件相关的明显的局部结构变异，这对于序列识别很重要。在复合物中，DNA 在蛋白质周围弯曲，蛋白质的 C 末端区域也发生了一些构象重排。来自自旋标记 DNA 的顺磁扰动实验、DNA 的化学位移扰动实验、先前的交叉饱和、蛋白质的化学位移扰动实验、突变分析信息和静电计算的结构约束已被用于使用已知的游离蛋白质溶液构象和有关 Mbp1:DNA 复合物的其他光谱信息产生详细的对接结构。基于蒙特卡罗的对接程序在完全溶剂化系统中采用受约束的 MD 进行，受到可用的实验约束，产生的模型可以解释可用的结构数据，并且可以合理化有关 Mbp1:DNA 复合物的广泛热力学数据。蛋白质：DNA 界面紧密堆积，并与少量特定接触相关。该结构显示出广泛的带正电荷的表面，这解释了聚电解质对结合的高贡献。
The solution structure of a 14 base-pair non-self complementary DNA duplex containing the consensus-binding site of the yeast transcription factor Mbp1 has been determined by NMR using a combination of scalar coupling analysis, time-dependent NOEs, residual dipolar couplings and C-13-edited NMR spectroscopy of a duplex prepared with one strand uniformly labeled with C-13-nucleotides. As expected, the free DNA duplex is within the B-family of structures, and within experimental limits is straight. However, there are clear local structural variations associated with the consensus CGCG element in the binding sequence that are important for sequence recognition. In the complex, the DNA bends around the protein, which also undergoes some conformational rearrangement in the C-terminal region. Structural constraints derived from paramagnetic perturbation experiments with spin-labeled DNA, chemical shift perturbation experiments of the DNA, previous cross-saturation, chemical shift perturbation experiments on the protein, information from mutational analysis, and electrostatics calculations have been used to produce a detailed docked structure using the known solution conformation of the free protein and other spectroscopic information about the Mbp1:DNA complex. A Monte Carlo-based docking procedure with restrained MD in a fully solvated system subjected to available experimental constraints produced models that account for the available structural data, and can rationalize the extensive thermodynamic data about the Mbp1:DNA complex. The protein:DNA interface is closely packed and is associated with a small number of specific contacts. The structure shows an extensive positively charged surface that accounts for the high polyelectrolyte contribution to binding.