NMR STRUCTURAL STUDIES OF CBFA RUNT DOMAIN DNA COMPLEX

CBFA Runt 结构域 DNA 复合体的 NMR 结构研究

• 批准号: 2887961
• 负责人: JOHN Hackett BUSHWELLER
• 金额: $ 13.46万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2887961
• 关键词: DNA binding protein; Drosophilidae; binding sites; dimer; genetic mapping; nuclear magnetic resonance spectroscopy; nucleic acid structure; protein sequence; protein structure; structural biology; transcription factor

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.

核心结合因子 (CBF) 最初被鉴定为 DNA 结合因子 特异性结合不对称序列 PyGPyGGT 的蛋白质， 对应于哺乳动物C型高度保守的“核心”位点 逆转录病毒增强子。 CBF 结合位点随后被 在许多 T 细胞特异性基因中发现，为 CBF 作为 T 细胞转录因子的作用。 额外的证据 因为 CBF 的重要性来自于小鼠体内这两个基因的敲除 这被证明是胚胎致死的，并且也被证明 造血发育受阻。 隔离及后续 CBF 的克隆表明该蛋白质是由 α亚基和β亚基。 α亚基与DNA接触 直接，而β亚基则不然，如缺乏所示 α 造成的磷酸盐接触数量的任何变化 贝塔的存在。 β 亚基与 α 亚基的结合 使 α 亚基对 DNA 的亲和力增加六倍，而无需 改变序列特异性。 α 亚基包含一个 128 与果蝇具有高度同源性的氨基酸区域 称为 Runt 的分割蛋白。 该域被称为 Runt 结构域已被证明负责这两个 DNA 和α亚基的β结合能力。 编码 CBF 亚基的四个基因中的两个通常是原癌基因 在人类白血病中被激活。 倒位和易位 这些基因中发现的与 30% 的 de novo 相关 人类急性髓性白血病。 CBF在白血病中的重要性 以及作为转录因子的正常作用 在分子水平上阐明其功能非常有趣 并且具有潜在的治疗作用。此外，缺乏任何 任一亚基与任何已知的结构基序的相似性使得它们 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 两者功能域的结构表征 CBF 的亚基以及相关的复合物。 我们是 使用核磁共振波谱法测定结构 CBFbeta(1-141) 以及 Runt 结构域-DNA 复合物。 本次活动的目的 建议确定 Runt 结构域-DNA 复合物的结构 并将 CBFbeta 结合位点映射到 Runt 域上。