Keniry M A, Banville D L, Simmonds P M, Shafer R
Research School of Chemistry, Australian National University, Canberra.
J Mol Biol. 1993 Jun 5;231(3):753-67. doi: 10.1006/jmbi.1993.1324.
A comprehensive two-dimensional 1H nuclear magnetic resonance spectral analysis of the ternary 4:2:1 mithramycin-Mg2+-d(A1C2C3C4G5G6G7T8)2 complex and the ternary 2:1:1 chromomycin-Mg(2+)-d(A21C2C3C4C4G5G6G7T8)2 complex is presented. The self-complementary oligonucleotide is found to bind two dimers of mithramycin in two identical off-center binding sites such that the twofold symmetry of the oligonucleotide is retained. In contrast, the same oligonucleotide binds only one dimer of chromomycin in a single but distinct off-center binding site. Two-dimensional nuclear Overhauser spectroscopy experiments show that the aglycone binding site of the drug dimer in each complex extends over almost four base-pairs and is similar in length to other complexes between chromomycin or mithramycin and oligonucleotides. The data demonstrate that the chromomycin dimer binding site is offset by one base-pair step from the dimer binding site in the mithramycin complex. This preferred binding site prevents two dimers of chromomycin binding to d(ACCCGGGT)2 for steric reasons and lends further support to previous work that showed the 5'-CG base-pair site is less favored by these drugs compared to the 5'GC and 5'-GG,5'-CC sites. Evidence is presented that suggests mithramycin may occupy either of two distinct binding sites on d(ACCCGGGT)2 when the drug concentration is not saturating. The nuclear magnetic resonance data demonstrate that the saccharide chains of this family of drugs do have a role in determining the binding site on nucleotides and as a consequence the CDE trisaccharide chain may alter its conformation to fulfil this role. Titration of mithramycin up to a drug-duplex ratio of 7:1 reveals further association of mithramycin with the complex but no new drug-oligonucleotide nuclear Overhauser enhancement contacts.
本文对4:2:1的光神霉素-Mg2+-d(A1C2C3C4G5G6G7T8)2三元复合物和2:1:1的嗜癌霉素-Mg(2+)-d(A21C2C3C4C4G5G6G7T8)2三元复合物进行了全面的二维1H核磁共振光谱分析。发现这种自我互补的寡核苷酸在两个相同的偏心结合位点结合两个光神霉素二聚体，从而保留了寡核苷酸的二重对称性。相比之下，相同的寡核苷酸在一个单一但不同的偏心结合位点仅结合一个嗜癌霉素二聚体。二维核Overhauser光谱实验表明，每种复合物中药物二聚体的糖苷配基结合位点延伸超过近四个碱基对，长度与嗜癌霉素或光神霉素与寡核苷酸之间的其他复合物相似。数据表明，嗜癌霉素复合物中二聚体结合位点相比，嗜癌霉素二聚体结合位点偏移了一个碱基对步长。这种优先结合位点由于空间位阻原因阻止了两个嗜癌霉素二聚体与d(ACCCGGGT)2结合，并进一步支持了先前的研究工作，即与5'GC和5'-GG、5'-CC位点相比，这些药物对5'-CG碱基对位点的偏好性较低。有证据表明，当药物浓度未饱和时，光神霉素可能占据d(ACCCGGGT)2上两个不同结合位点中的任何一个。核磁共振数据表明，这类药物的糖链在确定核苷酸上的结合位点方面确实起作用，因此CDE三糖链可能会改变其构象以发挥这一作用。将光神霉素滴定至药物-双链体比例为7:1，揭示了光神霉素与复合物的进一步结合，但没有新的药物-寡核苷酸核Overhauser增强接触。
