NMR Structural Studies of CBFB and CBFB-SMMHC

CBFB 和 CBFB-SMMHC 的 NMR 结构研究

• 批准号: 6747898
• 负责人: JOHN Hackett BUSHWELLER
• 金额: $ 25.9万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6747898
• 关键词: DNA; chimeric proteins; computer simulation; enzyme substrate complex; intermolecular interaction; model design /development; molecular dynamics; molecular site; nuclear magnetic resonance spectroscopy; physical model; protein structure function; protooncogene; structural biology; transcription factor

DESCRIPTION: 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 two of the four CBF genes in mice which were shown to be embryonic lethal and to have a profound blockage in hematopoietic development. Isolation and subsequent cloning of CBF showed the protein to be a heteromer consisting of a CBFA subunit and a CBFB subunit. The CBFA subunit contacts the DNA directly, whereas the CBFB subunit does not, as indicated by the lack of any changes in the number of phosphate contacts made by CBFA in the presence of CBFB. Binding of CBFB to CBFA increases the affinity of CBFA for DNA without altering the sequence specificity. The structures of the heterodimerization of CBFB and the DNA-binding domain, or Runt domain, of CBFA have been described by this lab. The mechanism by which CBFB enhances the binding of CBFA has not been established, thus specific aim #1 of this proposal is the determination of the structure of the heterodimerization domain of CBFB in the ternary complex with the Runt domain and DNA. This is part of Dr. Bushweller's overall goal of solving the structure of the ternary complex containing CBFB, CBFA, and DNA. 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 inv(16) involving the gene coding for CBFB produces a novel fusion protein with the functional domain of CBFB fused to the coiled-coil domain of a smooth muscle myosin protein. The mechanism of dysregulation caused by this oncoprotein has not been completely elucidated. In order to provide a structural basis for understanding the functioning of this oncoprotein form of CBFB, specific aim #2 proposes to solve the structure of a functional portion of the CBFB-SMMHC oncoprotein.

描述：核心结合因子（CBF）最初被确定为 DNA结合蛋白特异性结合与非对称序列 pygpyggt，对应于哺乳动物C型高度保守的“核心”位点 逆转录病毒增强剂。随后在A中鉴定了CBF结合位点 T细胞特异性基因的数量，提供了CBF作用的证据 T细胞转录因子。 CBF重要性的其他证据 来自小鼠四个CBF基因中两个的敲除，这些基因被证明是 胚胎致死，并在造血发育中遭受严重阻塞。 CBF的分离和后续克隆表明蛋白质为异构体 由CBFA亚基和CBFB亚基组成。 CBFA亚基联系 直接DNA，而CBFB亚基没有，如缺乏所示 CBFA在存在下进行的任何变化 CBFB。 CBFB与CBFA的结合增加了CBFA对无DNA的亲和力 改变序列特异性。异二聚体的结构 CBFB和CBFA的DNA结合域或Runt域已通过 这个实验室。 CBFB增强CBFA结合的机制尚未是 已建立，因此本提案的特定目的是确定 CBFB在三元络合物中的异二聚化结构域的结构 Runt域和DNA。这是布什威尔博士的总体目标的一部分 求解包含CBFB，CBFA和DNA的三元络合物的结构。 编码CBF亚基的四个基因中的两个通常是原始基因 在人类白血病中激活。在 这些基因与30％的从头急性髓细胞性白血病有关 人类。涉及CBFB基因编码的INV（16）产生一种新型融合 蛋白质具有CBFB的功能结构域，融合到A 平滑肌肌球蛋白蛋白。由此引起的失调机制 癌蛋白尚未完全阐明。为了提供一个 理解这种癌蛋白形式的功能的结构基础 CBFB，特定目标＃2提议解决功能部分的结构 CBFB-SMMHC癌蛋白。