Schroth G P, Kagawa T F, Ho P S
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331.
Biochemistry. 1993 Dec 14;32(49):13381-92. doi: 10.1021/bi00212a002.
We have solved the single-crystal X-ray structure of the complementary hexanucleotides d(m5-CGGGm5CG) and d(m5CGCCm5CG). The hexamer duplex was crystallized as Z-DNA, but contains a single C.G base pair that does not follow the alternating pyrimidine/purine rule for Z-DNA formation. This is the first crystal structure which serves to illustrate the structural consequences of placing a cytosine in the sterically disfavored syn conformation. In addition, since these sequences are not self-complementary, the individual strands of this asymmetric hexamer are unique in sequence and therefore distinguishable in the crystal lattice. Nevertheless, the crystal of this duplex is isomorphous with other Z-DNA hexamer structures. The asymmetry of this hexamer sequence required that the structure be solved using two unique models, which are distinguished by the orientation of hexanucleotides in the crystal lattice. In one model (the GG model) the cytosine in the syn conformation is packed against the terminal guanine base of a symmetry-related hexamer, while in the alternative model (the CC model) this cytosine sits exposed in a solvent channel of the lattice. We find that neither model alone can completely account for the observed electron densities. The two models ultimately were refined together. A composite structure consisting of 65% GG model and 35% CC model refined to an R-factor of 19.3%, which was significantly lower than refinements using either model alone. A detailed analysis of these two structures shows that, in spite of the out-of-alternation C.G base pair, the features characteristic of Z-DNA have been maintained. Both models, however, show significant local structural adjustments to accommodate the single cytosine base which is forced to adopt the syn conformation in each hexamer. In general, it appears that in order to relieve the energetically unfavorable steric contacts between the cytosine base in the syn conformation and the deoxyribose sugar, the base is forced into a highly buckled conformation, and that this large buckle in turn alters the conformation of neighboring residues. This unusual conformation also significantly weakens base-stacking interactions between the cytosine in syn and the adjacent residues in the helix and affects the exposure of the bases to solvent. We conclude that this crystal structure provides a molecular rationale for why nonalternating bases are energetically disfavored in Z-DNA.
我们解析了互补六核苷酸d(m5 - CGGGm5CG)和d(m5CGCCm5CG)的单晶X射线结构。该六聚体双链体以Z - DNA形式结晶，但包含一个不遵循Z - DNA形成的嘧啶/嘌呤交替规则的单一C.G碱基对。这是第一个用于说明将胞嘧啶置于空间不利的反式构象所产生的结构后果的晶体结构。此外，由于这些序列不是自我互补的，这个不对称六聚体的单链在序列上是独特的，因此在晶格中是可区分的。然而，这个双链体的晶体与其他Z - DNA六聚体结构是同晶型的。这个六聚体序列的不对称性要求使用两个独特的模型来解析结构，这两个模型通过晶格中六核苷酸的方向来区分。在一个模型（GG模型）中，反式构象的胞嘧啶与对称相关六聚体的末端鸟嘌呤碱基堆积在一起，而在另一个模型（CC模型）中，这个胞嘧啶暴露在晶格的溶剂通道中。我们发现单独一个模型都不能完全解释观察到的电子密度。最终将这两个模型一起进行了精修。由65%的GG模型和35%的CC模型组成的复合结构精修后的R因子为19.3%，这明显低于单独使用任何一个模型进行精修的结果。对这两个结构的详细分析表明，尽管存在非交替的C.G碱基对，Z - DNA的特征仍然得以保留。然而，两个模型都显示出显著的局部结构调整，以容纳每个六聚体中被迫采用反式构象的单个胞嘧啶碱基。一般来说，为了缓解反式构象的胞嘧啶碱基与脱氧核糖之间在能量上不利的空间接触，该碱基被迫进入高度弯曲的构象，而这种大的弯曲反过来又改变了相邻残基的构象。这种不寻常的构象也显著削弱了反式胞嘧啶与螺旋中相邻残基之间的碱基堆积相互作用，并影响了碱基对溶剂的暴露。我们得出结论，这个晶体结构为为什么非交替碱基在Z - DNA中在能量上不利提供了分子解释。
