NMR STRUCTURAL STUDIES OF DNA BINDING DOMAIN OF CBF?

CBF DNA 结合域的 NMR 结构研究？

• 批准号: 6309166
• 负责人: JOHN Hackett BUSHWELLER
• 金额: $ 0.75万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-15 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6309166
• 关键词: biomedical resource; hematology; human tissue; immunology; inflammation; neoplasm /cancer; nuclear magnetic resonance spectroscopy; nucleic acids; proteins

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.

核心结合因子（CBF）最初被确定为 特异性结合不对称序列的 DNA 结合蛋白 PyGPyGGT，对应于高度保守的“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 细胞转录的作用提供证据 因素。 对 CBF 作为 T 细胞转录作用的额外支持 最近，该因子来自造血功能被破坏的基因敲除小鼠。 早期发现被封锁。 隔离及后续 CBF 的克隆表明该蛋白质是由 ? 组成的异聚体。 和 ？亚基。 这 ？亚基直接接触DNA，而？ 子单位没有，正如缺乏任何变化所表明的那样 磷酸盐触点的数量？在存在？的情况下。装订 的 ？子单位到？亚基增加了 ? 的亲和力 DNA 亚基的六倍而不改变序列特异性。 这 ？亚基包含 128 个氨基酸区域，显示出高 与称为 Runt 的果蝇分割蛋白同源。 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 ? 的结合能力亚基。 四个基因中的两个 编码 CBF 亚基的原癌基因通常在人类中被激活 白血病。 这些基因中发现的倒位和易位 与人类 30% 的新发急性髓系白血病有关。 CBF 在白血病及其正常作用中的重要性 转录因子从分子水平阐明其功能 水平非常有趣并且具有潜在的治疗作用。 此外，Runt 域或 CBF 是否缺乏任何相似之处？ 任何已知的结构基序使它们成为重要的目标 结构测定。 我们工作的最终目标是 两个亚基相关域的结构表征 使用 NMR 分析 CBF。 该提案的目的是完整的 NMR Runt 的异核分配和结构测定 结构域-DNA复合物。 我们准备的 Runt 域结构是 176个氨基酸片段？亚基。 这被复杂化为 18 bp DNA 双链体以获得性能良好的蛋白质-DNA 复合物。 该复合物与 15N 标记的 Runt 的初步 15N-1H HSQC 光谱 域在 HSQC 谱中显示出非常低的色散，这需要 750 MHz 仪器待解决。 此外，我们还没有 此时，达到高于 0.7 mM 的浓度 复杂，因此还需要高磁场磁铁才能获得足够的 信噪比。 由于该复合物的大小 >30 kDa，我们有 选择用 50% 2H 以及 13C/15N 标记蛋白质 通过三重共振实验进行作业。 包含 2H 应该，如色氨酸阻遏物-DNA 复合物所示 例如，将相关 T2 增加到允许的值 记录分配过程的三重共振实验 以及用于结构测定的 NOESY 谱。