NMR STRUCTURAL STUDIES OF DNA BINDING DOMAIN OF CBF?

核心结合因子(CBF)最初被确定为 与不对称序列特异结合的DNA结合蛋白 PyGPyGGT，对应于在 哺乳动物C型逆转录病毒增强剂。CBF结合位点有 随后在一些T细胞特异性基因中被发现， CBF作为T细胞转录作用的证据 因素。CBF作为T细胞转录作用的额外支持 最近，因子来自基因敲除的小鼠，在这些小鼠中，造血功能 发现在早期阶段就被堵塞了。隔离和后续 CBF克隆表明该蛋白是一种异构体。 然后呢？亚单位。那个？亚基直接与DNA接触，而？ 子单元没有发生变化，这表明 制造的磷酸盐触点的数量？当着？的面。装订 那是什么？亚基到？亚基增加了？的亲和力 在不改变序列特异性的情况下，DNA亚基增加了六倍。 那个？亚基包含一个128个氨基酸区域，显示出高 与果蝇节段蛋白同源，称为Run。这是128 氨基酸结构域被称为Runt域。谷胱甘肽 仅含Run结构域的S-转移酶融合蛋白 显示该结构域负责DNA结合和 ？-绑定能力？亚单位。四个基因中的两个 编码CBF亚单位是人类中常见激活的原癌基因 白血病。这些基因中发现的倒位和易位 与人类30%的初治急性髓系白血病有关。 CBF在白血病中的重要性及其在正常情况下的作用 转录因子在分子水平上阐明其功能 这个水平非常有趣，而且有潜在的治疗作用。 此外，符文域或CBF没有任何相似之处吗？ 任何已知的结构主题使它们成为 结构确定。我们工作的最终目标是 两个亚基的相关结构域的结构特征 用核磁共振对脑血流量进行测定。这项提议的目的是完整的核磁共振 Run的异核指认和结构测定 结构域-DNA复合体。我们准备的Runt域构造是一个 该基因由176个氨基酸组成。亚单位。这是一个复杂的18 BPDNA双链，得到行为良好的蛋白质-DNA复合体。 该配合物与15N标记Run的15N-1H HSQC谱的初步研究 结构域在HSQC光谱中表现出很低的色散，这需要 750 MHz的仪器有待解决。此外，我们还没有，到 这一点，达到了浓度高于0.7毫米的这个 复杂，因此还需要高场磁铁才能获得足够的 信噪比。由于这个建筑群的30 kDa大小，我们有 选择用50%2H和13C/15N标记蛋白质 通过三重共振实验完成作业。这包括2H 如果，就像已经显示的色氨酸抑制物-DNA复合体一样， 例如，将相关的T2‘S增加到允许 作业过程中三重共振实验的记录 以及用于结构确定的NOESY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.