Structure determination of the closed dimeric conformation of TAp63α and investigation of its CK1 dependent activation

TAp63α 闭合二聚体构象的结构测定及其 CK1 依赖性激活的研究

• 批准号: 417339402
• 负责人: Professor Dr. Volker Dötsch
• 金额: --
• 依托单位: Institut für Biophysikalische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/417339402?language=en
• 关键词: Structure; determination; closed; dimeric; conformation

The special role of germ cells as the source for both somatic and germ cells of all following generations requires special quality control mechanisms. Oocytes are only generated during embryogenesis and female mammals are born with a finite number of oocytes that are arrested in prophase of meiosis I. This dictyate arrest starts after passing a check point that eliminates all oocytes that have not managed to repair the DNA double strand breaks that were introduced during the preceding process of homologous recombination. This checkpoint stays active in oocytes and is responsible for the elimination of oocytes suffering from DNA damage caused for example by chemotherapeutics or irradiation as part of a cancer therapy. Consequently, female cancer patients often become infertile and suffer from premature induction of menopause. Central player of this oocyte specific quality control system is the p53-homolog TAp63 alpha. In previous research we could show that TAp63 alpha adopts an autoinhibited, compact and only dimeric conformation in resting primary oocytes. Detection of DNA damage triggers a kinase cascade that results through phosphorylation by Chk2 and CK1 in the disruption of the autoinhibited structure and the formation of open and active tetramers that induce a pro-apoptotic transcriptional program. We have optimized a bacterial expression system for the production of the autoinhibited dimer. Based on mutational analysis and SAXS Measurements we have created a first model of the autoinhibited state and have shown that the C-terminal inhibitory domain together with the N-terminal transactivation domain forms a six-stranded β-sheet that blocks the tetramerization interface. We now want to determine the high resolution structure of TAp63α using cryo electronmicroscopy. Preliminary images of TAp63 alpha in negative stain mode show images of good quality suggesting that determining the structure by cryo EM is feasible. In addition we want to investigate the mechanism of phosphorylation dependent activation. So far we have shown that activation requires phosphorylation both by Chk2 and by CK1. Phosphorylation of a single serine residue in a loop preceding the C-terminal inhibitory domain by Chk2 recruits CK1 which adds four more phosphate groups. Electrostatic repulsion leads to the disruption of the inhibitory six-stranded beta-sheet. We want to determine the phosphorylation and activation kinetics using NMR spectroscopy and identify if CK1 uses a processive or distributive mode of phosphorylation. We have also started to investigate the interaction of p63 peptides with the kinase CK1 by determining a structure of CK1 in complex with a triple phosphorylated peptide and want to further investigate the interaction of differently phosphorylated peptides by x-ray crystallography and biophysical methods.

生殖细胞作为所有后代体细胞和生殖细胞来源的特殊作用需要特殊的质量控制机制。卵母细胞仅在胚胎发生过程中产生，雌性哺乳动物出生时具有有限数量的卵母细胞，这些卵母细胞在减数分裂i前期被阻止。这种决定性的阻止开始于通过一个检查点后，该检查点消除了所有未能修复同源重组过程中引入的DNA双链断裂的卵母细胞。这个检查点在卵母细胞中保持活跃，负责消除因化疗或放射治疗等癌症治疗引起的DNA损伤的卵母细胞。因此，女性癌症患者经常出现不孕和更年期提前的情况。这个卵母细胞特异性质量控制系统的核心参与者是p53同源物TAp63 α。在先前的研究中，我们可以发现TAp63 α在静止的原代卵母细胞中采用自抑制、致密且仅二聚体的构象。DNA损伤检测触发激酶级联反应，通过Chk2和CK1的磷酸化破坏自抑制结构，形成开放和活性四聚体，诱导促凋亡转录程序。我们已经优化了一种细菌表达系统，用于生产自抑制二聚体。基于突变分析和SAXS测量，我们创建了自抑制状态的第一个模型，并表明c端抑制结构域与n端反激活结构域一起形成六链β-片，阻断四聚化界面。我们现在想用低温电子显微镜确定TAp63α的高分辨率结构。在负染色模式下，TAp63 α的初步图像显示出良好的图像质量，表明用低温电镜测定其结构是可行的。此外，我们还想研究磷酸化依赖性激活的机制。到目前为止，我们已经证明激活需要Chk2和CK1的磷酸化。Chk2在c端抑制结构域前的环中磷酸化单个丝氨酸残基会招募CK1，从而增加四个磷酸基团。静电斥力导致抑制六链β -片的破坏。我们想确定磷酸化和活化动力学使用核磁共振波谱和确定是否CK1使用的是一个过程或分布模式的磷酸化。我们也已经开始研究p63多肽与CK1激酶的相互作用，通过确定CK1与三磷酸化肽复合物的结构，并希望通过x射线晶体学和生物物理方法进一步研究不同磷酸化肽的相互作用。