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原始癌基因的转录受到启动子位点的上游以及下游的多个顺式元素的调节。 CT启动子上游的一个顺式元素是CT元素。这是一个富含CT的序列，位于P1启动子上游的100个碱基。一种结合CT元件编码链的蛋白质是HNRNPK。 HNRNPK与CT元件的结合可上调C-MYC转录。 HNRNPK中有三个称为KH域的同源性重复序列。 HNRNPK蛋白的86个残基C末端段包含HNRNPK中的第三个KH基序（KH3）。除常规蛋白质NMR方法外，还使用液晶技术通过NMR光谱法确定了其三维结构。液晶环境在系统中产生一个轻微的秩序，重新引入了偶极耦合，从而提供有用的结构信息。使用偶极耦合信息计算得出的最终结构家族会导致0.17 Angstrom家族的根平方偏差（RMSD）。相反，在没有偶极耦合的情况下进行的计算的RMSD为0.32盎司。这些提供了对结构精度的定量评估，其中较小的RMSD值意味着更高的精度。此比较是在理想条件下进行的：所有NMR实验的良好信号与噪声，一些NMR实验重复了两次，以进行两次错误估计以及一致性检查，并且所有距离估计都保守地考虑了任何可能的系统错误。因此，对于在非理想条件下的典型NMR结构中，随着偶性偶联信息的添加，RMSD值的增加应该更大。与KH3结构确定并行，我们还使用偶极耦合信息开发了一种直接的结构细化方案。该协议正在提供给任何希望利用偶性耦合信息的实验室。还对KH3结构域进行了动态研究。在经历快速（PS）波动的KH3结构域中鉴定了一个柔性环（L52-R56）。与先前对FMR1类似KH结构域的研究相反，保守的循环（G30-G33）没有显示任何灵活性。为了通过CT元素更好地了解C-MYC调节，已经开始了一个平行的项目来确定细胞核酸结合蛋白（CNBP）的结构。该蛋白质结合与HNRNPK结合位点相反的链。 CNBP上调CT元素活动。已经制造了全长（176个残基）CNBP的结构。它由七个锌手指组成。 CNBP的最小构建体仍在保留核苷酸结合亲和力。我们已经完成了突变体（Gly26-ARG）KH3，野生型KH3和KH3+SS-DNA复合物的NMR主链动力学研究。我们已经表明，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。此时，我们可以访问DNA结合位点的所有动态参数和地图。我们目前正在比较所有这些参数，以根据结构和动态信息来表征KH3 SS-DNA相互作用。我们还使用KH3作为模型系统启动了有关侧链动力学的研究。我们已经开发了一个实验，可以在GLN和ASN的侧链上探测NH2部分。在自由和结合形式中对该NH2组动力学的比较将提供有关与SS-DNA目标的侧链相互作用的信息。该方法将扩展到蛋白质中的CH，CH2和CH3部分。去年，我们一直在尝试进行实验，以鉴定含KH3的蛋白质间域运动，并在HNRNPK的KH3侧链运动上完成我们的项目。为了研究域间的运动，我们使用了已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钙调蛋白。我们已经能够确认，为了量化慢速域运动，需要在不同的场强度下获得一系列NMR主链弛豫数据。此外，我们代表问题的一阶方法的简单模型似乎足以提供该运动的幅度和时间尺度。通过在钙调蛋白中对域运动进行空间模拟域运动，已经确认了估计的运动振幅。现在，我们正在应用这个简单的运动模型来研究KH3运动。 我们已经完善了可用于描述蛋白质中慢速Interam结构域运动的运动模型。我们的最新模型使用圆锥体中颤动的幻象近似，该锥体通过三个指数术语对内部运动进行了描述。到目前为止，我们的测试已经表明，当运动大（50度或更大）时，该模型是一个更合适的模型。在小幅度慢动作的极限中，较早的数学上更简单的模型就足够了。