Structural Studies of the c-myc Gene Regulation

人c-myc原癌基因的转录受启动子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。结合CT元件编码链的一种蛋白质是hnRNPk。HnRNPk与CT元件结合上调c-myc转录。HnRNPk中有三个同源重复。这些被称为KH域。HnRNPk蛋白的86个残基C-末端组成hnRNPk的第三个KH基序(Kh3)。用核磁共振波谱测定了hnRNPk蛋白在溶液中C-末端结构域的三维结构。除了常规的蛋白质核磁共振方法外，还用液晶技术测定了hnRNPk的Kh3结构。液晶环境在系统中产生了轻微的秩序。分子的有序性重新引入了偶极耦合，其中包含有用的结构信息。使用偶极耦合信息的添加计算的最终结构族导致0.17埃的族的均方根偏差(RMSD)。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估，RMSD值越小，精度越高。这种比较是在理想条件下进行的，即：对于所有的核磁共振实验都有良好的信噪比，为了误差估计以及一致性检查，一些核磁共振实验重复了两次，所有的距离估计都是保守的，以考虑到任何可能的系统误差。因此，对于典型的非理想条件下的核磁共振结构，随着偶极耦合信息的加入，RMSD值的增加应该要大得多。在确定Kh3结构的同时，我们还开发了一种利用偶极耦合信息进行结构精化的简单协议。该方案将提供给任何希望利用偶极耦合信息的实验室。还对HH3结构域进行了动态研究。开展动态研究有两个不同的目的。一种是获得关于分子整体翻滚以及局部微观运动的详细信息。二是利用松弛数据得到的流体动力学参数，对蛋白质结构的质量进行交叉检验。KH3的总体下落速度已被确定为6.87纳秒。在Kh3结构域中有一个柔性环(L52-R56)，它经历了快速(PS)的波动。与以前对FMR1类似KH结构域的研究相反，保守的环(G30-G33)没有显示出任何灵活性。这是因为进行这两项研究的pH值不同而引起的怀疑。这表明这种特殊的环路有可能发生化学交换，这种交换在进行Kh3研究的低pH值时会减弱。除了这一局部信息外，弛豫速率与KH3结构的总体拟合表明，该分子是非球形的，其长扩散轴与短扩散轴之比为1.37，不对称性(沿x轴和他们轴的扩散比)为1.12。我们指出，松弛数据对结构的拟合质量为结构本身提供了一个独立的质量评估。当动力学数据被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结构精确度提高了一倍。HnRNPk结构研究的下一步已经启动。构建了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结合常数也在调查中。此外，hnRNPk的C末端结构域含有3个SH3结合位点。一个含有Vav-SH3结构域的质粒正在构建中，以产生KH3结构域的靶肽。为了通过CT元件更好地了解c-myc的调控，已经启动了一个平行项目。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。该蛋白与CT元件的非编码链结合，从而与hnRNPk结合位点互补。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。目前正在探索一种仍然保持核苷酸结合亲和力的CNBP的最低结构。我们完成了突变型(Gly26-Arg)Kh3、野生型Kh3和Kh3+ss-DNA复合体的核磁共振骨架动力学研究。我们已经证明突变体KH3不具有ss-DNA结合活性。我们还对KH3+DNA络合物进行了滴定研究，以绘制DNA结合部位。在这一点上，我们可以访问所有的动态参数和DNA结合位点图。目前，我们正在比较所有这些参数，以基于结构和动态信息来表征KH3ss-DNA相互作用。我们还开展了以KH3为模型体系的侧链动力学研究。我们开展了一项实验，可以探测谷氨酰胺和天冬氨酸侧链上的氨基。比较自由和结合形式下NH2基团的动力学将提供关于侧链与ss-DNA靶标相互作用的信息。这一方法将被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.