Structure and dynamics of netropsin-poly(dA-dT).poly(dA-dT) complex: 500 MHz 1H NMR studies.
Structure and dynamics of netropsin-poly(dA-dT).poly(dA-dT) complex: 500 MHz 1H NMR studies.
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netropsin-poly(dA-dT).poly(dA-dT) 复合物的结构和动力学：500 MHz 1H NMR 研究。
DOI：
10.1080/07391102.1984.10507530
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发表时间：
1984
影响因子：
4.4
通讯作者：
Sarma,RH
中科院分区：
文献类型：
--
作者：
Gupta,G;Sarma,MH;Sarma,RH
已知抗生素netropsin特异性结合DNA中的A和T区域;结合模式是非嵌入的。显然，netropsin的质子供体与A的受体N3和T的受体O2之间的H-键合是一种很强的可能性，这可能导致这种特异性。在netropsin中，可能有8个质子供体：4个末端氨基和4个内部亚氨基。然而，末端氨基的甲基化并不改变netropsin与DNA的结合亲和力，但内部亚氨基的修饰显著降低了结合亲和力。因此，合乎逻辑的结论是，netropsin可以通过H键与A和T特异性相互作用，为了这样做，它应该从小沟接近螺旋。本文以poly（dA-dT）· poly（dA-dT）为模型体系，通过立体化学理论分析，观察到netropsin可以通过小沟与聚合物的-（T-A-T）序列形成特异性的B-键合。通过对自由poly（dA-dT）· poly（dA-dT）和netropsin-poly（dA-dT）· poly（dA-dT）复合物的~（31）P信号的监测，我们发现药物改变了DNA结构，使其从基本的单核苷酸重复变为非常显性的二核苷酸重复，但DNA-药物复合物中的碱基配对仍为Watson-Crick。通过在poly（dA-dT）· poly（dA-dT）存在下监测netropsin的亚氨基质子来判断氢键是否是特定的相互作用模式。实验在90%H_2O +10%D_2O中进行，采用分时长脉冲。结果发现，可交换的netropsin亚氨基质子出现在药物-DNA复合物中，并消失后增加的D2 O含量，从而证实，H-键合确实是具体的相互作用模式。根据这些和几个NOE测量，我们提出了一种poly（dA-dT）· poly（dA-dT）-netropsin复合物的结构。总之，实验数据表明netropsin通过形成特异性氢键与poly（dA-dT）· poly（dA-dT）结合，并且结合相互作用导致结构采用Watson-Crick配对二核苷酸重复基序。只有当药物从小沟接近DNA时，才能形成所提出的氢键。在NMR时间尺度内，配体与DNA之间的相互作用是快速的。从NOE实验数据来看，在netropsin存在下，poly（dA-dT）· poly（dA-dT）似乎以具有二核苷酸重复的右手和左手B-DNA双链体的平衡混合物存在，其中左手形式占优势。最后的结论是一个软的，因为它是非常困难的，以确保没有自旋扩散。在400个碱基对长的DNA双链体-药物复合物（如本研究中所用）中，右手螺旋和左手螺旋之间的平衡也可以意味着在同一分子中存在两个螺旋结构域，这些结构域之间快速交换或/和这些结构域沿着螺旋轴不受阻碍的运动/传播.
Antibiotic netropsin is known to bind specifically to A and T regions in DNA; the mode of binding being non-intercalative. Obviously, H-bonding between the proton donors of netropsin and acceptors N3 of A and 02 of T comes as a strong possibility which might render this specificity. In netropsin there could be 8 proton donors: four terminal amino groups and four internal imino groups. However, methylation of the terminal amino groups does not alter the binding affinity of netropsin to DNA—but the modification of the internal imino groups significantly lowers the binding affinity. Hence, the logical conclusion is that netropsin may specifically interact with A and T through H-bonding and in order to do so, it should approach the helix from the minor groove. The present paper provides experimental data which verify the conclusion mentioned above.Using poly(dA-dT)• poly(dA-dT) as a model system it was observed following a thorough theoretical stereochemical analysis that netropsin could bind to -(T-A-T) sequence of the polymer in the B-form through the minor groove by forming specific B-bonding. Models could be either right or left-handed B-DNA with a mono or dinucleotide repeat.By monitoring the31P signals of free poly(dA-dT) • poly(dA-dT) and netropsin-poly(dA-dT)• poly(dA-dT) complex we show that the drug changes the DNA structure from essentially a mononucleotide repeat to that of very dominant dinucleotide repeat; however the base- pairing in the DNA-drug complex remain to be Watson-Crick. Whether H-bonding is the specific mode of interaction was judged by monitoring the imino protons of netropsin in the presence of poly(dA-dT) • poly(dA-dT). This experiment was conducted in 90% H2O + 10% D2O Using the time-shared long pulse. It was found that exchangeable imino protons of netropsin appear in the drug-DNA complex and disappear upon increasing the D2O content; thus confirming that H-bonding is indeed the specific mode of interaction. From these and several NOE measurements, we propose a structure for poly(dA-dT)• poly(dA-dT(-netropsin complex.In summary, experimental data indicate that netropsin binds to poly(dA-dT)• poly(dA-dT) by forming specific hydrogen bonds and that the binding interaction causes the structure to adopt a Watson-Crick paired dinucleotide repeat motif. The proposed hydrogen bonds can form only if the drug approaches the DNA from the minor groove. Within the NMR time scale the interaction between the ligand and DNA is a fast one. From the NOE experimental data, it appears that poly(dA-dT)• poly(dA-dT) in presence of netropsin exists as an equilibrium mixture of right- and left-handed B-DNA duplexes with a dinucleotide repeat—with a predominance of the left-handed form. The last conclusion is a soft one because it was very difficult to make sure the absence of spin diffusion. In a 400 base pairs long DNA duplex- drug complex (as used in this study), equilibrium between right and left-handed helices can also mean the existence of both helical domains in the same molecule with fast interchange between these domains or/and unhindered motion/propagation of these domains along the helix axis.