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中有三个同源性重复。这些称为KH域。 HNRNPK蛋白的86个残基C末端段包含HNRNPK中的第三个KH基序（KH3）。溶液中HNRNPK蛋白的C末端结构域的三维结构已通过NMR光谱法确定。除常规蛋白质NMR方法外，还使用液晶技术确定了HNRNPK的Kh3结构。液晶环境在系统中产生略有秩序。分子的排序重新引入了包含有用的结构信息的偶极耦合。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为0.32盎司。 RMSD值对较小的RMSD值的结构的精度进行了定量评估。进行了比较，在理想条件下，即：所有NMR实验的良好信号与噪声的良好信号，对一些NMR实验进行了两次重复，以进行两次错误估计以及一致性检查，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，随着偶极耦合信息的添加，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制定了结构细化的简单方案。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。进行动态研究有两个不同的目的。一种是获取有关分子整体翻滚以及局部微观运动的详细信息。两个是使用从松弛数据得出的流体动力学参数来检查蛋白质结构的质量。 KH3的总体翻滚速率已确定为6.87 ns。 KH3域中有一个柔性环（L52-R56），它经历了快速（PS）波动。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。这是由于在pH中进行的，这是由于进行了两项研究的不同而怀疑的。因此，指出了这种特殊的循环进行化学交换的可能性，该化学交换会在进行KH3研究的情况下减少pH值。除了这些局部信息外，放松速率与KH3结构的总体拟合表明，该分子是非球形的，而长扩散轴与1.37的短扩散轴的比率是1.12的1.37和不对称性（沿X和它们轴的扩散比）。我们指出，放松数据对结构的拟合质量提供了对IT自我结构的独立质量评估。当将动态数据拟合到结构而没有偶极耦合的情况下，一个使用偶极精制结构时，卡方是1.5。这表明使用偶极耦合精制的结构是没有偶性耦合的结构的两倍。 HNRNPK结构研究的下一步已开始。 HNRNPK的Kh1+Kh2的构造已经制成。目前正在表征蛋白质表达。 KH1+Kh2结构域的单链DNA结合常数也正在研究中。另外，HNRNPK的C末端结构域包含3个SH3结合位点。正在构建一个包含VAV-SH3域的质粒，以生成Kh3Domain的靶肽。为了通过CT元素更好地了解C-MYC调节，已经启动了平行项目。在这个特定的项目中，我们想确定细胞核酸结合蛋白（CNBP）的结构。该蛋白质结合了CT元件的非编码链，因此将免费位点与HNRNPK结合位点结合。 CNBP上调CT元素活动。已经建立了全长（176个职业）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，umantKH3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thekh3 SS-DNA相互作用。我们还使用KH3AS A模型系统启动了对侧链动力学的研究。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组动态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。