RECOGNITION OF DNA BY THE E2 PROTEIN FROM PAPILLOMAVIRUS

乳头瘤病毒 E2 蛋白对 DNA 的识别

• 批准号: 2390384
• 负责人: JAMES D BALEJA
• 金额: $ 9.9万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2390384
• 关键词: DNA; DNA binding protein; Papillomavirus; computer simulation; intermolecular interaction; molecular dynamics; nuclear magnetic resonance spectroscopy; protein purification; protein structure; recombinant proteins; site directed mutagenesis; virus protein; DNA; DNA binding protein; Papillomavirus; computer simulation; intermolecular interaction; molecular dynamics; nuclear magnetic resonance spectroscopy; protein purification; protein structure; recombinant proteins; site directed mutagenesis; virus protein

The purpose of this investigation is to understand how the E2 protein from papillomavirus recognizes DNA. Three-dimensional structures will be determined using nuclear magnetic resonance (NMR) spectroscopy for the DNA-binding domain of bovine papillomavirus (BPV1) E2 protein, the DNA to which E2 protein binds, and mutants of the protein. The approach will be to: l, Purify recombinant forms of the protein; 2, Optimize conditions for NMR spectroscopy; 3, Obtain NMR data for the DNA-binding domain; 4, Study site-specific mutants of E2; 5, Obtain NMR data for the binding-site DNA; 6, Determine structures from the NMR data; 7, Measure dynamics in the protein; and 8, Perform molecular modeling studies on the structures determined. The DNA-binding domain encompasses the most C-terminal 101 residues (310-410) of bovine papillomavirus E2 protein. This includes a 16 amino acid residue segment that has not been studied before structurally and which clearly appears to be important for E2 protein function. A comparison of structures for the previously published protein-DNA complex to that of the unbound protein and unbound DNA will reveal the conformational changes that the protein and DNA must undergo in order to recognize each other. Furthermore, dynamics within the protein will be studied by amide proton exchange and relaxation time measurements to understand the role of flexibility in the E2 protein-DNA recognition process. An important feature of this application is the study of site- specific mutants of E2 protein. Although many of these mutants have been characterized as to their DNA-binding and transactivating ability little is known about the structural consequences of introducing the amino acid change. Alteration of structure caused by replacement of the cysteine in the DNA recognition helix will be examined in particular since the DNA- binding affinity of E2 may be regulated by modification of cysteinyl sulfur through a novel reduction/oxidation mechanism. The papillomaviruses are a small family of DNA viruses that cause epithelial tumors in a variety of vertebrate hosts. The genetics and biochemistry of bovine papillomavirus (BPV1) have been most extensively characterized. The human (HPV) and bovine viruses have many properties in common, including homologous DNA-binding domains of E2 protein. E2 plays essential roles in both viral DNA replication and viral gene expression and therefore regulates viral growth and transformation of the host cell. In humans the vast majority of cervical carcinomas contain HPV viral DNA. A better understanding of the way in which E2 regulates viral activity is an important first step in the development of novel methods to correct aberrant processes in cellular control such as those associated with cancer caused by the human virus.

这项研究的目的是了解如何从 乳头瘤病毒识别DNA。三维结构将是 使用核磁共振（NMR）光谱测定 牛乳头瘤病毒（BPV1）E2蛋白的DNA结合结构域，DNA至 E2蛋白结合了蛋白质的突变体。方法将是 到：l，纯化蛋白质的重组形式； 2，优化条件 NMR光谱； 3，获得DNA结合域的NMR数据； 4，研究 E2的位点特异性突变体； 5，获得结合位点DNA的NMR数据； 6，从NMR数据确定结构； 7，测量动力学 蛋白质; 8，对结构进行分子建模研究 决定。 DNA结合域涵盖了最多的C末端101 牛乳头瘤病毒E2蛋白的残基（310-410）。这包括16 氨基酸残基段，在结构上尚未研究 这显然对于E2蛋白功能很重要。一个 比较先前发表的蛋白-DNA复合物的结构 对于未结合的蛋白质和未结合的DNA的DNA将揭示 蛋白质和DNA必须经历的构象变化才能 互相认识。此外，蛋白质内的动力学将是 由酰胺质子交换和放松时间测量到 了解灵活性在E2蛋白DNA识别中的作用 过程。该应用的一个重要特征是研究站点 - E2蛋白的特异性突变体。尽管这些突变体中有许多已经 其DNA结合和反式激活能力的特征很少 已经知道引入氨基酸的结构后果 改变。通过替代半胱氨酸引起的结构的改变 DNA识别螺旋将特别检查，因为DNA- E2的结合亲和力可以通过Cysteinyl的修饰来调节 通过新颖的还原/氧化机制硫。乳头瘤病毒 是在A中引起上皮肿瘤的一小部分DNA病毒 各种脊椎动物宿主。牛的遗传学和生物化学 乳头瘤病毒（BPV1）的表征最广泛。人类 （HPV）和牛病毒具有许多共同特性，包括 E2蛋白的同源DNA结合结构域。 E2在 病毒DNA复制和病毒基因表达，因此 调节宿主细胞的病毒生长和转化。在人类中 绝大多数宫颈癌含有HPV病毒DNA。更好 了解E2调节病毒活性的方式是一种 开发新方法以纠正的重要第一步 细胞控制中的异常过程，例如 人类病毒引起的癌症。