NMR structural and kinetic characterization of a homeodomain diffusing and hopping on nonspecific DNA
NMR structural and kinetic characterization of a homeodomain diffusing and hopping on nonspecific DNA
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DOI：
10.1073/pnas.0605868103
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发表时间：
2006-10-10
影响因子：
11.1
通讯作者：
Clore, G. Marius
中科院分区：
文献类型：
--
作者：
Iwahara, Junji;Zweckstetter, Markus;Clore, G. Marius
非特异性蛋白质-DNA相互作用是固有的动态的，并且涉及蛋白质沿着DNA的扩散和蛋白质从一个DNA分子或片段跳跃到另一个。理解基因调控蛋白如何在结构和动力学方面与DNA非特异性相互作用是具有挑战性的，因为实验的可观察量是许多快速交换状态的集合平均值。通过使用各种NMR光谱技术，包括弛豫分析，顺磁弛豫增强，和残余偶极耦合，我们的特点是结构和动力学方面的HoxD 9同源结构域与非特异性，24-bp的DNA双链体在一个系统中的蛋白质不限于任何特定的网站的相互作用。数据显示，HoxD 9以与在特异性复合物中观察到的相同的结合模式和取向与非特异性DNA结合。然而，与DNA接触的Arg侧链的流动性在非特异性复合物中相对于特异性复合物增加。两种不同的非特异性DNA分子之间的分子间易位的动力学也已被分析，并揭示在高DNA浓度（如那些存在于体内）直接从一个非特异性复合物转移到另一个非特异性DNA分子发生，而不通过游离蛋白质的中介。这一发现提供了一种简单的机制，通过允许在蛋白质从一个片段跳到另一个片段时更有效地对可用DNA位点进行采样，来加速在非特异性位点的海洋中体内对特异性位点的靶搜索。
Nonspecific protein-DNA interactions are inherently dynamic and involve both diffusion of the protein along the DNA and hopping of the protein from one DNA molecule or segment to another. Understanding how gene regulatory proteins interact nonspecifically with DNA in terms of both structure and dynamics is challenging because the experimental, observables are an ensemble average of many rapidly exchanging states. By using a variety of NMR spectroscopic techniques, including relaxation analysis, paramagnetic relaxation enhancement, and residual dipolar couplings, we have characterized structural and kinetic aspects of the interaction of the HoxD9 homeodomain with a nonspecific, 24-bp DNA duplex in a system in which the protein is not constrained to any particular site. The data reveal that HoxD9 binds to nonspecific DNA with the same binding mode and orientation as that observed in the specific complex. The mobility, however, of Arg side-chains contacting the DNA is increased in the nonspecific complex relative to the specific one. The kinetics of intermolecular translocation between two different nonspecific DNA molecules have also been analyzed and reveal that at high DNA concentrations (such as those present in vivo) direct transfer from one nonspecific complex to another nonspecific DNA molecule occurs without going through the intermediary of free protein. This finding provides a simple mechanism for accelerating the target search in vivo for the specific site in a sea of nonspecific sites by permitting more effective sampling of available DNA sites as the protein jumps from one segment to another.