Structural Studies Of The C-myc Gene Regulation

C-myc 基因调控的结构研究

• 批准号: 6541683
• 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/6541683
• 关键词: apoptosis; binding proteins; calmodulin; chemical models; cytosine; dipole moment; genetic regulation; genetic regulatory element; intermolecular interaction; 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-element upstream from the c-myc promoters is the CT element. This is a CT rich sequence which is located 100 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 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 this has been determined by NMR spectroscopy using the liquid crystal technique in addition to the conventional protein NMR method. The liquid crystal environment produces a slight order in the system which reintroduces dipolar coupling providing 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, calculations carried out without the dipolar couplings has an RMSD of 0.32 Angstrom. These provide a quantitative evaluation of the precision of the structures with the smaller RMSD value implying higher precision. This comparison was made under ideal conditions: good signal to noise for all of the NMR experiments, some NMR experiments repeated twice for error estimates as well as consistency check, and all distance estimates 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 straight forward 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. One flexible loop (L52-R56) was identified in the KH3 domain which undergoes rapid (ps) fluctuation. In contrast to a previous study of a similar KH domain of FMR1 the conserved loop (G30-G33) does not show any flexibility. In order to get a better understanding of c-myc regulation through the CT element, a parallel project has been initiated to determine the structure of the cellular nucleic acid binding protein (CNBP). This protein binds the strand opposite to the hnRNPk binding site. CNBP upregulates the CT element activity. A construct of the full length (176 residues) 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 mutant KH3. We have also done titration studies on the KH3+DNA complex to map the DNA binding site. At this point, we have access to all dynamic parameters and maps of the DNA binding site. We are currently comparing all of these parameters to characterize the KH3 ss-DNA interaction based on structure as well as dynamic information. We also have initiated a study on side chain dynamics using KH3 as a model system. We have developed an experiment where we can probe the NH2 moiety on the side chain of Gln and Asn. Comparison of the dynamics 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, and CH3 moieties in the protein. This last year we have been trying to carry out experiments to identify inter-domain motions in KH3-containing protein as well as to finish our project on the side chain motion in KH3 of hnRNPk. In order to study the inter-domain motions we have used a model system that has been studied extensively to test our analysis model. The model system chosen was a calcium binding protein, calmodulin. We have been able to confirm that in order to quantify slow inter-domain motions a series of NMR backbone relaxation data obtained at different field strengths would be required. Furthermore, our simple model that represents a first order approach to the problem, seems to be adequate to provide the amplitude as well as the time scale of this motion. The estimated amplitude of motion has been confirmed by sterically modeling the domain motion in calmodulin. We are now applying this simple motional model to study KH3 motion. We have refined the motional model that can be used to describe slow internam domain motions in protein. Our latest model use a Pade approximation of wobbled in a cone which results in a description of the internal motion by three exponential terms. So far our tests have shown this model to be a more suitable model when the motion is large (angle of inflection of 50 degrees or larger). In the limit of small amplitude slow motion the earlier and mathematically simpler model is sufficient.

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，称为 KH 结构域。 hnRNPk 蛋白的 86 个残基 C 端片段包含 hnRNPk 中的第三个 KH 基序 (KH3)。除了传统的蛋白质NMR方法之外，还使用液晶技术通过NMR光谱测定了其三维结构。液晶环境在系统中产生轻微的秩序，重新引入偶极耦合，提供有用的结构信息。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。这些提供了结构精度的定量评估，RMSD 值越小意味着精度越高。这种比较是在理想条件下进行的：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，并且所有距离估计都保守地进行，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直接的结构细化协议。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。在 KH3 结构域中发现了一个柔性环 (L52-R56)，该环经历快速 (ps) 波动。与之前对 FMR1 的类似 KH 结构域的研究相比，保守环 (G30-G33) 没有表现出任何灵活性。为了更好地了解 CT 元件对 c-myc 的调节，我们启动了一个平行项目来确定细胞核酸结合蛋白 (CNBP) 的结构。该蛋白结合与 hnRNPk 结合位点相对的链。 CNBP 上调 CT 元素活性。已构建出全长（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骨架动态研究。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。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 基团的动力学将提供侧链与 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。去年，我们一直在尝试进行实验来鉴定含 KH3 的蛋白质中的域间运动，并完成我们关于 hnRNPk 的 KH3 中的侧链运动的项目。为了研究域间运动，我们使用了一个经过广泛研究的模型系统来测试我们的分析模型。选择的模型系统是钙结合蛋白，即钙调蛋白。我们已经能够确认，为了量化缓慢的域间运动，需要在不同场强下获得的一系列 NMR 主干弛豫数据。此外，我们的简单模型代表了解决该问题的一阶方法，似乎足以提供该运动的幅度和时间尺度。估计的运动幅度已通过对钙调蛋白中的域运动进行立体建模得到证实。我们现在应用这个简单的运动模型来研究 KH3 运动。 我们改进了运动模型，可用于描述蛋白质中缓慢的内部结构域运动。我们的最新模型使用圆锥体摆动的 Pade 近似，从而通过三个指数项来描述内部运动。到目前为止，我们的测试表明，当运动较大（拐角为 50 度或更大）时，该模型是更合适的模型。在小幅度慢运动的限制下，早期且数学上更简单的模型就足够了。