Albright R A, Mossing M C, Matthews B W
Howard Hughes Medical Institute and Department of Physics, University of Oregon, Eugene 97403, USA.
Protein Sci. 1998 Jul;7(7):1485-94. doi: 10.1002/pro.5560070701.
The structure has been determined at 3.0 A resolution of a complex of engineered monomeric Cro repressor with a seven-base pair DNA fragment. Although the sequence of the DNA corresponds to the consensus half-operator that is recognized by each subunit of the wild-type Cro dimer, the complex that is formed in the crystals by the isolated monomer appears to correspond to a sequence-independent mode of association. The overall orientation of the protein relative to the DNA is markedly different from that observed for Cro dimer bound to a consensus operator. The recognition helix is rotated 48 degrees further out of the major groove, while the turn region of the helix-turn-helix remains in contact with the DNA backbone. All of the direct base-specific interactions seen in the wild-type Cro-operator complex are lost. Virtually all of the ionic interactions with the DNA backbone, however, are maintained, as is the subset of contacts between the DNA backbone and a channel on the protein surface. Overall, 25% less surface area is buried at the protein DNA interface than for half of the wild-type Cro-operator complex, and the contacts are more ionic in character due to a reduction of hydrogen bonding and van der Waals interactions. Based on this crystal structure, model building was used to develop a possible model for the sequence-nonspecific interaction of the wild-type Cro dimer with DNA. In the sequence-specific complex, the DNA is bent, the protein dimer undergoes a large hinge-bending motion relative to the uncomplexed form, and the complex is twofold symmetric. In contrast, in the proposed nonspecific complex the DNA is straight, the protein retains a conformation similar to the apo form, and the complex lacks twofold symmetry. The model is consistent with thermodynamic, chemical, and mutagenic studies, and suggests that hinge bending of the Cro dimer may be critical in permitting the transition from the binding of protein at generic sites on the DNA to binding at high affinity operator sites.
已在3.0埃分辨率下确定了工程化单体Cro阻遏蛋白与一个七碱基对DNA片段复合物的结构。尽管DNA序列与野生型Cro二聚体每个亚基所识别的共有半操纵子一致，但晶体中由分离的单体形成的复合物似乎对应于一种不依赖序列的结合模式。蛋白质相对于DNA的总体取向与结合到共有操纵子的Cro二聚体所观察到的明显不同。识别螺旋进一步从大沟中旋转出48度，而螺旋-转角-螺旋的转角区域仍与DNA主链接触。野生型Cro-操纵子复合物中所有直接的碱基特异性相互作用都消失了。然而，与DNA主链的几乎所有离子相互作用都得以保留，DNA主链与蛋白质表面一个通道之间的接触子集也是如此。总体而言，蛋白质-DNA界面处掩埋的表面积比野生型Cro-操纵子复合物一半的情况少25%，并且由于氢键和范德华相互作用的减少，接触更具离子性质。基于此晶体结构，利用模型构建来开发野生型Cro二聚体与DNA序列非特异性相互作用的可能模型。在序列特异性复合物中，DNA弯曲，蛋白质二聚体相对于未结合形式经历大的铰链弯曲运动，并且复合物具有二重对称性。相比之下，在所提出的非特异性复合物中，DNA是直的，蛋白质保留类似于无配体形式的构象，并且复合物缺乏二重对称性。该模型与热力学、化学和诱变研究一致，并表明Cro二聚体的铰链弯曲可能在允许从蛋白质在DNA上的一般位点结合转变为在高亲和力操纵子位点结合方面起关键作用。
