Structural Studies of the c-myc Gene Regulation

c-myc 基因调控的结构研究

• 批准号: 6432658
• 负责人: NICO TJANDRA
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432658
• 关键词: apoptosis; binding proteins; cytosine; dipole moment; genetic regulation; genetic regulatory element; genetic transcription; mathematical model; molecular dynamics; nuclear magnetic resonance spectroscopy; protein binding; protein structure function; protooncogene

The transcription of the human c-myc proto-oncogene is regulated by multiple cis-elements upstream as well as downstream of the promoter sites. One cis-elements upstream from the c-myc promoters is the CT element. This is a CT rich sequence which is located100 bases upstream from the P1 promoter. One protein that binds the coding strand of the CT element is hnRNPk. Binding of hnRNPk to the CT element upregulates c-myc transcription. There are three homology repeats in hnRNPk. These are called the KH domains. The 86 residue C-terminal segment of the hnRNPk protein comprises the third KH motif (KH3) in hnRNPk. The three-dimensional structure of the C-terminal domain of hnRNPk protein in solution has been determined by NMR spectroscopy. The structure of KH3 of hnRNPk was determined using the liquid crystal technique in addition to conventional protein NMR method. The liquid crystal environment produces a slight order in the system. The ordering of the molecule reintroduces dipolar coupling which contain useful structural information. The final family of structures calculated using the addition of dipolar coupling information results in root mean square deviation (RMSD)for the family of 0.17 Angstrom. In contrast, calculation carried out without the dipolar couplings has an RMSD of 0.32 Angstrom. The RMSD value provides a quantitative evaluation of the precision of the structures with smaller RMSD value being higher precision. This comparison was made under ideal conditions, i.e. : good signal to noise for all of the NMR experiments, some NMR experiments were repeated twice for error estimates as well as consistency check, all distance estimates were done conservatively to take into account any possible systematic error. Thus for a typical NMR structure under non-ideal conditions the increase of the RMSD value with the addition of dipolar coupling information should be much greater. In parallel to the KH3 structure determination we also developed a straightforward protocol of structure refinement using the dipolar coupling information. This protocol is being provided to any laboratory which wishes to take advantage of dipolar coupling information. A dynamic study of KH3 domain has also been carried out. There are two different purposes for carrying out the dynamic study. One is to get the detail information regarding the overall tumbling of the molecule as well as the local microscopic motion. Two is to use the hydrodynamic parameters derived from the relaxation data to cross check the quality of protein structure. The overall tumbling rate of the KH3 has been determined to be 6.87 ns. There is one flexible loop (L52-R56) in the KH3 domain which undergoes rapid (ps) fluctuation. In contrast to previous study of a similar KH domain of FMR1 the conserved loop (G30-G33) do not show any flexibility. This is suspected due to the different in pH under which the two studies were carried out. Thus points to the possibility of this particular loop undergoing chemical exchange which diminishes at low pH where the KH3 study was done. In addition to this local information the overall fit of the relaxation rates to the KH3structure has revealed that the molecule is non spherical with the ratio of the long diffusion axis to the short diffusion axis of 1.37 and the asymmetry (ratio of diffusion along the x and they axis) of 1.12. We pointed out that the quality of the fit of the relaxation data to the structure provides an independent quality assessment of the structure it self. When the dynamics data were fitted to the structure without the dipolar coupling one observed a residual error (chi squared) of 3.0 while using the dipolar refined structure the chi-squared is 1.5. This is suggesting that the structure refined using dipolar coupling is twice as accurate as the one without dipolar coupling. The next steps in the structural study of hnRNPk has been initiated. The construct of KH1+KH2 of hnRNPk has been made. The protein expression is currently being characterized. Single stranded DNA binding constant for the KH1+KH2 domain is also under investigation. In addition the C-terminal domain of hnRNPk contains 3 SH3 binding sites. A plasmid containing the Vav-SH3domain is being constructed to produce the target peptide for KH3domain.In order to get a better understanding of c-myc regulation through the CT element a parallel project has been initiated. In this particular project we would like to determine the structure of cellular nucleic acid binding protein (CNBP). This protein binds the non-coding strand of the CT element, thus the complimentary site to the hnRNPk binding site. CNBP upregulates the CT element activity. A construct of the full length (176residues) CNBP has been made. It consists of seven zinc fingers. A minimum construct of CNBP which still retains the nucleotide binding affinity is being probed. We have completed the NMR backbone dynamic studies of mutant(Gly26-Arg) KH3, wild type KH3, and KH3+ss-DNA complex. We have shown that there is no ss-DNA binding activity for the mutantKH3. We have also done titration study on the KH3+DNA complex to map the DNA binding site. At this point we have access to all dynamic parameters and map of the DNA binding site. We are currently comparing all of these parameters to characterize theKH3 ss-DNA interaction based on structure as well as dynamic information. We also have initiated a study on side chain dynamics using KH3as a model system. We have developed an experiment where we can probe NH2 moiety on the side chain of Gln and Asn. Comparison of the dynamic of this NH2 group in the free and bound form would provide information on side chain interaction with the ss-DNA target. This methodology will be extended to look at CH, CH2, andCH3 moieties in the protein.

人c-myc原癌基因的转录受到启动子位点上游和下游的多个顺式元件的调节。 c-myc 启动子上游的一个顺式元件是 CT 元件。这是一个富含 CT 的序列，位于 P1 启动子上游 100 个碱基处。一种结合 CT 元件编码链的蛋白质是 hnRNPk。 hnRNPk 与 CT 元件的结合上调 c-myc 转录。 hnRNPk 中有 3 个同源重复。这些被称为 KH 域。 hnRNPk 蛋白的 86 个残基 C 端片段包含 hnRNPk 中的第三个 KH 基序 (KH3)。溶液中 hnRNPk 蛋白 C 端结构域的三维结构已通过 NMR 光谱确定。除了传统的蛋白质NMR方法外，还使用液晶技术测定了hnRNPk的KH3结构。液晶环境在系统中产生轻微的秩序。分子的排序重新引入了偶极耦合，其中包含有用的结构信息。使用添加偶极耦合信息计算出的最终结构族导致该族的均方根偏差 (RMSD) 为 0.17 埃。相比之下，在没有偶极耦合的情况下进行的计算的 RMSD 为 0.32 埃。 RMSD值提供了结构精度的定量评价，RMSD值越小，精度越高。该比较是在理想条件下进行的，即：所有 NMR 实验都有良好的信噪比，一些 NMR 实验重复两次以进行误差估计和一致性检查，所有距离估计均保守地进行，以考虑任何可能的系统误差。因此，对于非理想条件下的典型 NMR 结构，随着偶极耦合信息的添加，RMSD 值的增加应该要大得多。在确定 KH3 结构的同时，我们还使用偶极耦合信息开发了一种简单的结构细化协议。该协议正在提供给任何希望利用偶极耦合信息的实验室。 KH3结构域的动态研究也已进行。进行动态研究有两个不同的目的。一是获取有关分子整体翻滚以及局部微观运动的详细信息。二是使用从弛豫数据导出的流体动力学参数来交叉检查蛋白质结构的质量。 KH3 的整体翻滚速率已确定为 6.87 ns。 KH3结构域中有一个柔性环（L52-R56），它经历快速（ps）波动。与之前对 FMR1 类似 KH 结构域的研究相比，保守环 (G30-G33) 没有表现出任何灵活性。这可能是由于两项研究进行时的 pH 值不同所致。因此表明这个特定的环路可能发生化学交换，在 KH3 研究进行的地方，化学交换在低 pH 值下会减弱。除了该局部信息之外，弛豫率与 KH3 结构的整体拟合表明，该分子是非球形的，长扩散轴与短扩散轴的比率为 1.37，且不对称性（沿 x 和 x 方向的扩散比率）它们的轴）为 1.12。我们指出，松弛数据与结构的拟合质量提供了结构本身的独立质量评估。当将动力学数据拟合到没有偶极耦合的结构时，观察到残余误差（卡方）为 3.0，而使用偶极精化结构时，卡方为 1.5。这表明使用偶极耦合精修的结构比没有偶极耦合的结构精度高一倍。 hnRNPk 结构研究的后续步骤已经启动。已经构建了hnRNPk的KH1+KH2。目前正在对蛋白质表达进行表征。 KH1+KH2 结构域的单链 DNA 结合常数也在研究中。此外，hnRNPk 的 C 端结构域包含 3 个 SH3 结合位点。正在构建包含 Vav-SH3 结构域的质粒，以产生 KH3 结构域的目标肽。为了更好地了解通过 CT 元件对 c-myc 的调节，已经启动了一个平行项目。在这个特定的项目中，我们想要确定细胞核酸结合蛋白（CNBP）的结构。该蛋白结合 CT 元件的非编码链，因此是 hnRNPk 结合位点的互补位点。 CNBP 上调 CT 元素活性。已经构建了全长（176 个残基）CNBP。它由七个锌指组成。目前正在探索仍保留核苷酸结合亲和力的 CNBP 最小构建体。我们已经完成了突变型(Gly26-Arg) KH3、野生型KH3和KH3+ss-DNA复合物的NMR骨架动力学研究。我们已经证明突变体 KH3 没有 ss-DNA 结合活性。我们还对 KH3+DNA 复合物进行了滴定研究，以绘制 DNA 结合位点图谱。此时，我们可以访问 DNA 结合位点的所有动态参数和图谱。我们目前正在比较所有这些参数，以基于结构和动态信息来表征 KH3 ss-DNA 相互作用。我们还启动了一项使用 KH3 作为模型系统的侧链动力学研究。我们开发了一项实验，可以探测 Gln 和 Asn 侧链上的 NH2 部分。比较游离和结合形式的 NH2 基团的动态将提供侧链与 ss-DNA 靶标相互作用的信息。该方法将扩展到研究蛋白质中的 CH、CH2 和 CH3 部分。