Structural Studies of the c-myc Gene Regulation

人类c-myc原癌基因的转录受到启动子位点上游和下游多个顺式元件的调控。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。结合CT元件编码链的一种蛋白质是hnRNPk。hnRNPk与CT元件结合可上调c-myc转录。hnRNPk有三个同源重复序列。这些被称为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）。用核磁共振光谱法测定了溶液中hnRNPk蛋白c端结构域的三维结构。在常规蛋白质核磁共振法的基础上，利用液晶技术测定了hnRNPk的KH3结构。液晶环境在系统中产生轻微的秩序。分子的有序重新引入了偶极耦合，其中包含有用的结构信息。加入偶极耦合信息计算的最终结构族的均方根偏差（RMSD）为0.17埃。相反，在没有偶极耦合的情况下进行的计算的RMSD为0.32埃。RMSD值可以定量评价结构的精度，RMSD值越小，精度越高。这种比较是在理想条件下进行的，即：所有核磁共振实验的信噪比都很好，一些核磁共振实验重复两次进行误差估计和一致性检查，所有距离估计都是保守的，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核磁共振结构，加入偶极耦合信息后RMSD值的增加应该大得多。在确定KH3结构的同时，我们还利用偶极耦合信息开发了一种简单的结构改进方案。本协议提供给任何希望利用偶极耦合信息的实验室。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。一是获得分子整体翻滚和局部微观运动的详细信息。二是利用弛豫数据得到的水动力参数对蛋白质结构质量进行交叉检验。测定了KH3的总翻滚速率为6.87 ns。在KH3结构域中有一个柔性环路（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，保守环（G30-G33）没有表现出任何灵活性。这是由于两项研究的pH值不同而引起的怀疑。因此指出了这种特殊的循环进行化学交换的可能性，这种交换在KH3研究完成的低pH值下减少。除了这些局部信息外，弛豫率与kh3结构的整体拟合表明分子是非球形的，长扩散轴与短扩散轴的比值为1.37，不对称（沿x轴和它们轴的扩散比）为1.12。我们指出松弛数据与结构的拟合质量为结构本身的质量提供了一个独立的评价。当动力学数据拟合到没有偶极耦合的结构时，观察到残差（卡方）为3.0，而使用偶极精细化结构时，卡方为1.5。这表明使用偶极耦合改进的结构比没有偶极耦合的精确两倍。hnRNPk结构研究的下一步已经开始。制备了hnRNPk的KH1+KH2结构。目前正在对该蛋白的表达进行表征。KH1+KH2结构域的单链DNA结合常数也在研究中。此外，hnRNPk的c端结构域含有3个SH3结合位点。一个含有vav - sh3结构域的质粒正在被构建以产生kh3结构域的靶肽。为了更好地理解通过CT元件的c-myc调控，一个平行项目已经启动。在这个特殊的项目中，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元件活性。构建了全长（176个残基）的CNBP。它由七个锌指组成。一个最小的CNBP结构仍然保留核苷酸结合亲和力正在探索。我们完成了突变体（Gly26-Arg） KH3、野生型KH3和KH3+ss-DNA复合物的核磁共振骨架动力学研究。我们已经证明突变体kh3没有ss-DNA结合活性。我们还对KH3+DNA复合体进行了滴定研究，绘制了DNA结合位点。至此，我们获得了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，以表征基于结构和动态信息的kh3 ss-DNA相互作用。我们还利用kh3作为模型系统对侧链动力学进行了研究。我们已经开发了一个实验，我们可以探测Gln和Asn侧链上的NH2部分。比较游离态和结合态NH2基团的动态变化，可以提供与ss-DNA靶标侧链相互作用的信息。该方法将扩展到观察蛋白质中的CH， CH2和ch3部分。

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.