NMR STRUCTURAL STUDIES OF DNA BINDING DOMAIN OF CBF?

核心结合因子（CBF）最初被鉴定为一种 特异性结合不对称序列的DNA结合蛋白 PyGPyGGT，对应于PyGPyGGT中高度保守的"核心"位点， 哺乳动物C型逆转录病毒增强子。 CBF结合位点具有 随后在许多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提供了证据， 因子 对CBF作为T细胞转录的作用的额外支持 因子最近来自基因敲除小鼠，在这些小鼠中， 发现在早期阶段被阻塞。 隔离和随后的 CBF的克隆表明，该蛋白质是一个异聚体组成的一个？ 然后呢？亚单位 什么？亚基直接接触的DNA，而？ 亚基没有，如缺乏任何变化所示， 磷酸盐触点数量由？当着……的面结合 的？亚单位的？亚基增加的亲和力？ 在不改变序列特异性的情况下， 什么？亚基含有128个氨基酸的区域，显示高的 与果蝇称为Runt的分节蛋白同源。 这128 氨基酸结构域被称为Runt结构域。 谷胱甘肽 仅具有Runt结构域的S-转移酶（GST）融合蛋白具有 显示该结构域负责DNA结合和 ?-的约束力？亚单位 四个基因中的两个 编码CBF亚单位的原癌基因通常在人类中被激活， 白血病 在这些基因中发现的倒位和易位 与30%的人类新发急性髓性白血病有关。 CBF在白血病中的重要性以及其作为一种 转录因子在分子水平上阐明了其功能 非常有趣，并且有潜在的治疗价值。 此外，缺乏任何相似的侏儒域或CBF？ 任何已知的结构基序使它们成为 结构测定 我们工作的最终目标是 两个亚基的相关结构域的结构表征 CBF的NMR。 该提案的目的是完整的NMR Runt异序分配与结构判定 结构域-DNA复合物。 我们准备的Runt结构域构建体是一个 176个氨基酸的片段？亚单位 这是复杂的18 bp DNA双链体，以获得表现良好的蛋白质-DNA复合物。 该复合物与15N标记Runt的初步15N-1H HSQC光谱 域在HSQC光谱中显示出非常低的色散，这需要 一个750兆赫的仪器来解决。 此外，我们还没有， 在这一点上，达到了高于0.7 mM的浓度， 复杂，因此还需要高场磁体以获得足够的磁场。 信噪比 由于该复合物的> 30 kDa大小，我们 选择用50%2H以及13C/15N标记蛋白质， 通过三重共振实验分配。 包含2H 应该，正如已经显示的trp阻遏物-DNA复合物， 例如，将相关T2增加到允许 分配过程的三重共振实验记录 以及用于结构测定的NOESY光谱。

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 support for CBF's role as a T-cell transcription factor has recently come from knockout mice in which hematopoiesis was found to be blocked at an early stage. Isolation and subsequent cloning of CBF showed the protein to be a heteromer consisting of an ? and ? subunit. The ? subunit contacts the DNA directly, whereas the ? subunit does not, as indicated by the lack of any changes in the number of phosphate contacts made by ? in the presence of ?. Binding of the ? subunit to the ? subunit increases the affinity of the ? subunit for the DNA sixfold without altering the sequence specificity. The ? subunit contains a 128 amino acid region displaying a high homology to the Drosophila segmentation protein called Runt. This 128 amino acid domain is referred to as the Runt domain. Glutathione S-Transferase (GST) fusion proteins with the Runt domain alone have shown this domain is responsible for both the DNA-binding and ?-binding capabilities of the ? 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% 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 make elucidation of its function at the molecular level extremely interesting and potentially therapeutically useful. In addition, the lack of any resemblance of the Runt domain or CBF? to any known structural motifs makes them important targets for structure determination. The ultimate objective of our work is the structural characterization of the relevant domains of both subunits of CBF using NMR. The aim of this proposal is the complete NMR heteronuclear assignment and structure determination of a Runt domain-DNA complex. The Runt domain construct we have prepared is a 176 amino acid fragment of the ? subunit. This is complexed to an 18 bp DNA duplex to obtain a well-behaved protein-DNA complex. Preliminary 15N-1H HSQC spectra of this complex with 15N-labeled Runt domain showed very low dispersion in the HSQC spectrum which required a 750 MHz instrument to be resolved. Additionally, we have not, to this point, reached concentrations higher than 0.7 mM for this complex, thus also requiring a high field magnet to obtain adequate signal-to-noise. Due to the >30 kDa size of this complex, we have chosen to label the protein with 50% 2H as well as 13C/15N for assignments via triple resonance experiments. This inclusion of 2H should, as has been shown for the trp repressor-DNA complex for example, increase the relevant T2's to values that permit the recording of triple resonance experiments for the assignment process as well as NOESY spectra for structure determination.