NMR studies of restriction enzyme-DNA interactions: Role of conformation in sequence specificity
NMR studies of restriction enzyme-DNA interactions: Role of conformation in sequence specificity
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DOI：
10.1021/bi0473758
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发表时间：
2005-04-05
期刊：
影响因子：
2.9
通讯作者：
Dupureur, CM
中科院分区：
文献类型：
--
作者：
Dupureur, CM
序列特异性DNA结合蛋白被认为在与靶（同源）和非靶（非同源）序列结合时采用不同的构象。有生物化学和晶体学的证据表明，这种行为是重要的介导序列识别的Mg（II）依赖型11限制性内切酶。尽管如此，很少有系统的比较这些酶在各种复合物的结构行为。在这里，H-1-N-15 HSQC NMR光谱应用于PvuII核酸内切酶（2 × 18 kDa），以更好地理解序列识别和酶构象行为之间的关系。在不存在和存在金属离子的情况下收集的游离酶的光谱表明，虽然有一个适度的骨架构象响应后结合Ca（II），这不会发生与Mg（II）。底物结合本身伴随着与大规模构象响应一致的非常显著的光谱变化。HSQC光谱的酶绑定到同源（特异性）和非同源（非特异性）寡核苷酸的存在下的Ca（II）是显着不同的，揭示了第一次的PvuII同源复合物在溶液中的结构独特性。NMR光谱重叠和晶体学数据（C-α rmsd）之间的强相关性允许将非特异性Pvull复合物表征为比特异性复合物更类似于游离酶。总的来说，这些数据支持这样一种观点，即是DNA而不是金属离子促进了酶的独特构象反应。因此，金属离子在复合物形成中的主要作用是驱动底物亲和力和稳定性，而不是构象引发酶的底物结合和序列识别。这些结果不仅提供了有价值的见解蛋白质-DNA相互作用的机制，但也证明了这些代表性的核酸系统的结构-功能研究的NMR光谱的效用。
Sequence specific DNA binding proteins are thought to adopt distinct conformations when binding to target (cognate) and nontarget (noncognate) sequences. There is both biochemical and crystallographic evidence that this behavior is important in mediating sequence recognition by the Mg(II)-dependent type 11 restriction enzymes. Despite this, there are few systematic comparisons of the structural behavior of these enzymes in various complexes. Here, H-1-N-15 HSQC NMR spectroscopy is applied to PvuII endonuclease (2 x 18 kDa) in an effort to better understand the relationship between sequence recognition and enzyme conformational behavior. Spectra of the free enzyme collected in the absence and presence of metal ions indicate that while there is a modest backbone conformational response upon binding Ca(II), this does not occur with Mg(II). Substrate binding itself is accompanied by very dramatic spectral changes consistent with a large-scale conformational response. HSQC spectra of the enzyme bound to cognate (specific) and noncognate (nonspecific) oligonucleotides in the presence of Ca(II) are dramatically distinct, revealing for the first time the structural uniqueness of a PvuII cognate complex in solution. The strong correlation between NMR spectral overlap and crystallographic data (C-alpha rmsd) permits characterization of the nonspecific Pvull complex as being more similar to the free enzyme than to the specific complex. Collectively, these data support the notion that it is the DNA, not the metal ion, which promotes a unique conformational response by the enzyme. It therefore follows that the principle role of metal ions in complex formation is one of driving substrate affinity and stability rather than conformationally priming the enzyme for substrate binding and sequence recognition. These results not only provide valuable insights into the mechanism of protein-DNA interactions but also demonstrate the utility of NMR spectroscopy in structure-function studies of these representative nucleic acid systems.