- 學位論文
MDC1與CHK2中 FHA結構域在DNA損傷反應之結構與功能研究
Structural and Functional Study of FHA Domain of MDC1 and CHK2 in DNA Damage Response
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摘要
此論文著重在DNA損傷反應中，MDC1與CHK2結合結構的解析。MDC1蛋白在DNA損傷反應中藉由與許蛋白在DNA損傷反應中藉由與許多蛋白交互作用，扮演起一個傳遞者的角色。MDC1蛋白不僅可以與ATM及MRE11這種感應蛋白結合(sensor proteins)，也可以與像CHK2這種作用蛋白(effector proteins)結合。細胞會透過複雜的磷酸化訊息傳遞來感應、誘發及調控DNA損傷反應，進而讓細胞進行DNA修復、細胞週期停滯或者細胞凋亡。先前的研究顯示MDC1-FHA結構域會與CHK2的第68個磷酸化賴氨酸結合，此位置也可以與其本身的FHA結構域結合，讓CHK2形成雙體化而活化。為了瞭解其分子機制，我們解出MDC1-FHA結構域及其結合CHK2 第68個磷酸化賴氨酸胜肽的原子結構。顯然地，無論在溶液或晶體中MDC1-FHA結構域皆形成雙體的構型。結構及蛋白結合分析都支持MDC1-FHA對於pThr+3配位體的專一性，也理解MDC1如何與CHK2結合。原子結構提供MDC1-FHA雙體結合介面的資訊，因此我們挑選了破懷雙體卻還保有與磷酸化賴氨酸結合能力的MDC1-FHA突變體來進行雙體功能的研究。免疫共沉澱法及split-GFP分析顯示這些MDC1蛋白的突變體在細胞內無法形成雙體。在MDC1蛋白knockdown的細胞中，這些突變體對於輻射比較敏感。結果顯示在DNA損傷的位置，必須由MDC1蛋白的雙體構型來調控其蛋白的動態平衡，才能正確地傳遞DNA損傷的訊號。實驗結果暗示MDC1的雙體結構可能扮演著超級鷹架的角色去與DNA損傷有關的蛋白結合。另外， 雙體化後的自我磷酸化是激酶活化的普遍機制。在DNA損傷後，ATM激酶會使CHK2激酶的第68個賴氨酸磷酸化，接著CHK2激酶本身的FHA結構域會與磷酸化的賴氨酸結合，使CHK2雙體化，進而讓其T-loop自我磷酸化來讓CHK2激酶活化。然而，整個活化過程的分子機制還不是很清楚。因此，我們利用native chemical ligation的方式生產出第68個賴氨酸磷酸化的CHK2激酶，然後結合分析型超高速離心與小角度X光散射來瞭解其生物物理特性與結構。結果顯示CHK2激酶的第68個賴氨酸磷酸化在穩定CHK2雙體中扮演很重要的角色，而小角度X光散射的結果也說明了此雙體化可能讓兩個激酶處在正確的位置，使T-loop的自我磷酸化更有效率。
並列摘要
This work focuses on the structural basis of the FHA domain (forkhead-associated domain) in the interaction between MDC 1 (mediator of DNA damage checkpoint 1) and CHK2 (checkpoint kinase 2) upon DNA damage. The MDC1 protein functions as a key mediator that interacts with multiple proteins involved in DNA damage response (DDR) pathway. It binds to not only sensor proteins like ATM and MRE11, but also effector proteins such as CHK2. The complicated phospho-signaling network controls the sensing, initiating, and mediating steps that lead to downstream repair pathways, cell-cycle checkpoints, and apoptosis. Previously, the CHK2 binding site for MDC1-FHA was shown to be pThr68 (the same site recognized by the FHA domain of CHK2 for dimerization and activation of CHK2). To elucidate the molecular mechanism of MDC1-CHK2 interaction, we solved crystal structures of mouse MDC1-FHA and its complex with a human CHK2 peptide containing pThr68. Surprisingly, MDC1-FHA exists as an intrinsic dimer in solution and in crystals. Structural and binding analyses support the pThr+3 ligand specificity of FHA domains, and provide structural insight into MDC1-CHK2 interaction. In order to test whether the dimerization of MDC1-FHA directs MDC1 function in vivo, we selected different MDC1-FHA mutants with disrupted dimerization while maintaining the pThr-binding ability. The full-length MDC1 protein containing such mutations not only failed to dimerize in vivo as suggested by split-GFP system, but also failed to rescue cellular radio-sensitivity caused by MDC1 knockdown. In addition, our result shows that the dimeric feature affects the MDC1 protein turnover rate on DNA lesion sites by which the accurate DNA damage signal can be executed. It implies that the dimeric feature may play a role of super-scaffold to interact with other proteins after DNA damage. In addition, dimerization-dependent trans autophophorylation is a common mechanism to active kinase. Activated ATM kinase phosphorylates CHK2 on Thr68 to trigger CHK2 activation in DNA damage. The pThr68 interacts with its FHA domain, leading to dimerization and T-loop autophosphorylation. However, the molecular basis of this activation process remains unclear. Here we use site-specifically pThr68 CHK2 for biophysical characterization by AUC and provide structural explanation by SAXS analyses. The results show that pThr68 plays a critical role to stabilize CHK2 dimerization. The SAXS results show that pThr68-mediated dimerization is possible to bring two kinases to correct orientation for efficient activation loop trans autophosphorylation.
參考文獻
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