Brown D R, Kurz M, Kearns D R, Hsu V L
Department of Chemistry, University of California, San Diego, La Jolla 92093-0342.
Biochemistry. 1994 Jan 25;33(3):651-64. doi: 10.1021/bi00169a005.
The solution conformations of a DNA oligomer and its complexes with the anticancer drug actinomycin D (ActD) were characterized using homo- and heteronuclear NMR techniques. Previous high-resolution NMR investigations of ActD-DNA complexes employed symmetric double-stranded DNA oligomers, yielding two identical symmetry-related complexes. In order to understand the important effects that neighboring base pairs and/or unusual nucleic acid structures may have on ActD binding specificity and orientation, we chose to study the oligonucleotide d(TCGCGTTTTCGCGA), which adopts a hairpin structure in solution. NOE cross-peak intensities were used to generate distance constraints for molecular dynamics simulations and structure determinations of the free oligonucleotide and for both complexes. A total of 86 intermolecular NOEs were identified for each complex, 27 of which involve exchangeable protons. These intermolecular NOEs along with changes in the phosphorus chemical shifts were used to determine the drug binding site on the DNA. As expected, ActD intercalated exclusively at the single d(GC) step in the DNA hairpin. Interestingly, although the two complexes, which differ by the orientation with which the asymmetric drug chromophore intercalates the DNA, were not formed in equal concentrations, their conformations are very similar. The RMS difference of the DNA hairpin in the two complexes is only 1.10 A. The structures of the minor groove binding pentapeptide rings are not affected by any of the changes in the normal double-helical structure imposed by the hairpin loop. The total pairwise RMS difference over all atoms for the four peptides (two per complex) in the calculated structures is 0.72 A. Conversely, the structure of the hairpin loop is not appreciably changed upon binding--the RMS difference between the free DNA loop region and the loop region in the two complexes is 1.68 A and only 0.43 A between the two complexes. Our data also support a possible conformation of the d(T)4 loop that does not possess a thymine-thymine "wobble" base pair.
使用同核和异核核磁共振技术对一种DNA寡聚物及其与抗癌药物放线菌素D（ActD）的复合物的溶液构象进行了表征。先前对ActD-DNA复合物的高分辨率核磁共振研究使用了对称双链DNA寡聚物，产生了两个相同的对称相关复合物。为了了解相邻碱基对和/或异常核酸结构可能对ActD结合特异性和方向产生的重要影响，我们选择研究寡核苷酸d（TCGCGTTTTCGCGA），它在溶液中呈发夹结构。NOE交叉峰强度用于生成分子动力学模拟的距离约束以及游离寡核苷酸和两种复合物的结构测定。每种复合物共鉴定出86个分子间NOE，其中27个涉及可交换质子。这些分子间NOE以及磷化学位移的变化用于确定药物在DNA上的结合位点。正如预期的那样，ActD仅插入DNA发夹中的单个d（GC）步骤。有趣的是，尽管两种复合物因不对称药物发色团插入DNA的方向不同而形成的浓度不相等，但其构象非常相似。两种复合物中DNA发夹的均方根偏差仅为1.10 Å。小沟结合五肽环的结构不受发夹环施加的正常双螺旋结构变化的影响。计算结构中四个肽（每个复合物两个）所有原子的总成对均方根偏差为0.72 Å。相反，发夹环的结构在结合后没有明显变化——游离DNA环区域与两种复合物中环区域之间的均方根偏差为1.68 Å，两种复合物之间仅为0.43 Å。我们的数据还支持d（T）4环的一种可能构象，该构象不具有胸腺嘧啶-胸腺嘧啶“摆动”碱基对。
