NMR STRUCTURAL STUDIES OF CBFA RUNT DOMAIN DNA COMPLEX

核心结合因子(CBF)最初被认为是一种DNA结合因子 与不对称序列PyGPyGGT特异结合的蛋白质， 与哺乳动物C型高度保守的“核心”部位相对应 逆转录病毒增强剂。CBF结合位点随后被 在一些T细胞特异性基因中被鉴定，为 CBF作为T细胞转录因子的作用。其他证据 因为CBF的重要性来自于小鼠这两个基因的敲除 它们被证明是胚胎致死的，也被证明是 在造血发育方面有障碍。隔离和后续 CBF的克隆表明该蛋白是一个异构体，由一个 Alpha亚基和Beta亚基。阿尔法亚单位与DNA接触 直接，而β亚基不直接，如缺乏所示 中的磷酸盐触点数量的任何变化 贝塔的存在。β亚基与α亚基的结合 将阿尔法亚单位与DNA的亲和力增加六倍 改变序列特异性。阿尔法亚单位包含一个128 与果蝇高度同源的氨基酸区域 一种叫做Run的分段蛋白。此域称为 矮小结构域，并已被证明对这两个DNA负责 和α亚基的β结合能力。 编码CBF亚基的四个基因中有两个通常是原癌基因 在人类白血病中被激活。倒位和易位 在这些基因中被发现与30%的新生相关 人类的急性髓系白血病。脑血流量在白血病中的重要性 以及作为转录因子的正常作用 在分子水平上阐明它的功能非常有趣 而且有潜在的治疗作用。此外，缺乏任何 任何一个亚基与任何已知结构基序的相似性使它们 结构确定的重要目标。 我们工作的总体目标是彻底的生物物理和 两者功能结构域的结构表征 CBF的亚基以及所涉及的相关复合体。我们是 用核磁共振波谱测定化合物的结构 CBFβ(1-141)以及Run结构域-DNA复合体。这样做的目的是 建议确定Run结构域-DNA复合体的结构 并将CBFbeta结合位点映射到Run结构域上。

Core binding factor (CBF) was originally identified as a DNA-binding protein that specifically binds to the asymmetric sequence PyGPyGGT, corresponding to the highly conserved "core" site in mammalian type C retrovirus enhancers. CBF binding sites have subsequently been identified in a number of T cell specific genes, providing evidence for the role of CBF as a T-cell transcription factor. Additional evidence for the importance of CBF has come from knockouts of both genes in mice which were shown to be embryonic lethal and which also were shown to have blockage in hematopoietic development. Isolation and subsequent cloning of CBF showed the protein to be a heteromer consisting of an alpha subunit and a beta subunit. The alpha subunit contacts the DNA directly, whereas the beta subunit does not, as indicated by the lack of any changes in the number of phosphate contacts made by alpha in the presence of beta. Binding of the beta subunit to the alpha subunit increases the affinity of the alpha subunit for DNA sixfold without altering the sequence specificity. The alpha subunit contains a 128 amino acid region displaying a high homology to the Drosophila segmentation protein called Runt. This domain is referred to as the Runt domain and has been shown to be responsible for the both the DNA and beta-binding capabilities of the alpha subunit. Two of the four genes encoding CBF subunits are proto-oncogenes commonly activated in human leukemias. The inversion and translocations identified in these genes are associated with 30 percent of de novo acute myeloid leukemias in humans. The importance of CBF in leukemia as well as in its normal role as a transcription factor makes elucidation of its function at the molecular level extremely interesting and potentially therapeutically useful. In addition, the lack of any resemblance of either subunit to any known structural motifs makes them important targets for structure determination. The overall objective of our work is the thorough biophysical and structural characterization of the functional domains of the two subunits of CBF as well as the relevant complexes involved. We are employing NMR spectroscopy for the structure determinations of CBFbeta(1-141) as well as a Runt domain-DNA complex. The aims of this proposal are to determine the structure of the Runt domain-DNA complex and map the CBFbeta binding site on the Runt domain.