Physical and structural basis for the strong interactions of the -ImPy- central pairing motif in the polyamide f-ImPyIm
Physical and structural basis for the strong interactions of the -ImPy- central pairing motif in the polyamide f-ImPyIm
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DOI：
10.1021/bi061245c
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发表时间：
2006-11-14
期刊：
影响因子：
2.9
通讯作者：
Lee, Moses
中科院分区：
文献类型：
--
作者：
Buchmueller, Karen L.;Bailey, Suzanna L.;Lee, Moses
聚酰胺f-ImPyIm对其同源DNA的亲和力高于母体类似物偏端霉素A（10倍）或结构异构体f-PyImIm（250倍）对其相应同源DNA序列的亲和力。这些发现导致了双字母聚酰胺“语言”的形成，其中- ImPy-中心配对与沃森-克里克DNA的关联比- PyPy-、- PyIm-和- ImIm-更强。在这里，我们进一步表征f-ImPyIm和f-PyImIm，我们报告热力学和结构之间的差异- ImPy-（f-ImPyIm）和-PyIm-（f-PyImIm）中心配对。DNA酶I足迹法研究证实，f-ImPyIm是一种比偏端霉素A和f-PyImIm更强的结合剂，并且f-ImPyIm优先结合CGCG超过多个竞争序列。f-ImPyIm和f-PyImIm与其同源序列结合的差异得到DNA熔解研究的Na+依赖性的支持，其中需要显著更高的Na+浓度来匹配f-ImPyIm稳定CGCG的能力与f-PyImIm稳定CCGG的能力。通过圆二色性和等温滴定量热法测试了f-ImPyIm在四碱基CGCG识别位点之外的选择性，这表明f-ImPyIm对（A中心点T）CGCG（A中心点T）的选择性超过（G中心点C）CGCG（G中心点C）。此外，邻近该6 bp结合位点的变化不影响f-ImPyIm亲和力。量热研究显示，f-ImPyIm、f-PyImIm和偏端霉素A与它们各自的发夹同源序列的结合是放热的;然而，焓、熵和热容（Δ Cp）的变化对每种三酰胺形成2：1复合物的贡献不同。f- ImPyIm与CGCG结合的Δ Cp的实验和理论测定结果吻合良好（分别为-142和-177 cal mol（-1）K-1）. f-ImPyIm和f-PyImIm与它们各自的同源DNA复合的1H-1 NMR证实了不同的2：1复合物的正合作形成。NMR结果还表明，这些三酰胺结合在DNA小沟，寡核苷酸保留B-型构象。使用NMR实验的最小距离限制，分子建模和动力学被用来说明f-ImPyIm和CGCG之间的结构互补性。总的来说，NMR和ITC实验表明，2：1 f-ImPyIm-CGCG复合物的形成实现了比2：1 f-PyImIm-CCGG复合物的结构更有序和更有利的结构。
The polyamide f- ImPyIm has a higher affinity for its cognate DNA than either the parent analogue, distamycin A ( 10-fold), or the structural isomer, f-PyImIm ( 250-fold), has for its respective cognate DNA sequence. These findings have led to the formulation of a two-letter polyamide " language" in which the - ImPy- central pairings associate more strongly with Watson- Crick DNA than - PyPy-, - PyIm-, and - ImIm-. Herein, we further characterize f-ImPyIm and f-PyImIm, and we report thermodynamic and structural differences between - ImPy- ( f-ImPyIm) and -PyIm- ( f-PyImIm) central pairings. DNase I footprinting studies confirmed that f-ImPyIm is a stronger binder than distamycin A and f-PyImIm and that f-ImPyIm preferentially binds CGCG over multiple competing sequences. The difference in the binding of f-ImPyIm and f-PyImIm to their cognate sequences was supported by the Na+-dependent nature of DNA melting studies, in which significantly higher Na+ concentrations were needed to match the ability of f-ImPyIm to stabilize CGCG with that of f-PyImIm stabilizing CCGG. The selectivity of f-ImPyIm beyond the four- base CGCG recognition site was tested by circular dichroism and isothermal titration microcalorimetry, which shows that f-ImPyIm has marginal selectivity for ( A center dot T) CGCG( A center dot T) over ( G center dot C)CGCG( G center dot C). In addition, changes adjacent to this 6 bp binding site do not affect f-ImPyIm affinity. Calorimetric studies revealed that binding of f-ImPyIm, f-PyImIm, and distamycin A to their respective hairpin cognate sequences is exothermic; however, changes in enthalpy, entropy, and heat capacity ( Delta Cp) contribute differently to formation of the 2:1 complexes for each triamide. Experimental and theoretical determinations of Delta Cp for binding of f- ImPyIm to CGCG were in good agreement (- 142 and - 177 cal mol(-1) K-1, respectively). H-1 NMR of f-ImPyIm and f-PyImIm complexed with their respective cognate DNAs confirmed positively cooperative formation of distinct 2:1 complexes. The NMR results also showed that these triamides bind in the DNA minor groove and that the oligonucleotide retains the B- form conformation. Using minimal distance restraints from the NMR experiments, molecular modeling and dynamics were used to illustrate the structural complementarity between f-ImPyIm and CGCG. Collectively, the NMR and ITC experiments show that formation of the 2:1 f-ImPyIm-CGCG complex achieves a structure more ordered and more thermodynamically favored than the structure of the 2:1 f-PyImIm-CCGG complex.