FHA結構域蛋白質之結構及功能研究

Structural and Functional Study of FHA Domain Proteins

翁睿宏(Weng, Jui Hung)

指導教授 ： 蔡明道 ； 余靖

國立清華大學/理學院/化學系/博士(2015年)

摘要

FHA結構域是唯一具專一性識別磷酸蘇氨酸的結構域。許多參與調控信號複合體和連接信號傳導途徑的多結構域蛋白質包含了FHA結構域。FHA結構域具有多種功能並且在廣泛的生化反應中扮演重要的角色。本篇論文主要著重在兩個FHA結構域蛋白：Rad53和TIFA 。(一) Rad53是人類細胞週期檢查點激酶 Chk2在芽殖酵母裡的同源蛋白。Rad53 參與了第一間期，第二間期和合成期的細胞週期檢查點。 Rad53的自磷酸化調控Rad53的激酶活性，而二聚化調節了Rad53的自磷酸化。利用X射線晶體學，我們解析了Rad532-466 (包含了SCD1、FHA1和激酶結構域：Rad53SFK)的結構並發現了打開和關閉兩種激酶結構域的形式。這兩種形式的不同在於激酶結構域N瓣相對於Cα螺旋的移動和旋轉。在關閉的形式中，Cα螺旋比較靠近3β鏈，在Cα螺旋上的穀胺酸244和β3鏈上的離胺酸227形成鹽橋。而這個離胺酸227對於和三磷酸腺苷的結合與穩定相當重要。此外X射線小角散射實驗發現了Rad53SFK在溶液中有多種結構。磷酸化Rad53SFK的FHA結構域和激酶結構域則相當靠近以及穩定。此研究結果對於Rad53的自磷酸化機制以及磷酸結合所誘導的二聚化調節提供了重要的見解。並顯示了二聚化驅動含濃度誘導機制的不同。 (二) TIFA參與細胞內核因子活化B細胞κ輕鏈增強子(NF-κB)的活化。 當細胞受到刺激時，TIFA的蘇氨酸9會被磷酸化，而磷酸化的TIFA會聚合。聚合的TIFA會促進腫瘤壞死因子受體相關因子6(TRAF6)聚合和泛素化(ubiquitination)，進而激活NF-κB。本實驗所解析的TIFA FHA結構域結構和TIFA 與 磷酸化蘇氨酸9的TIFA N端胜肽1-15 （ pThr9 - TIFA1-15 ）複合物結構，展示了TIFA 獨特的 FHA結構域結構和新的FHA結構域結合模式，以及TIFA二聚體不同的二聚體界面。由所解析的結構可推論，TIFA磷酸化蘇氨酸和FHA結構域的交互作用是發生於不同的二聚體之間，不是發生於二聚體內。此研究讓我們了解TIFA二聚體結構以及聚合發生的分子機制。

關鍵字

FHA 結構域

並列摘要

Forkhead-associated (FHA) domain is the only signaling domain that recognizes phosphothreonine (pThr) specifically. The FHA domain is often part of the multi-domain domain proteins that facilitates the assembling of signaling complex and connecting of the signaling pathway. It has diverse functions in a protein and plays major roles in jodyad53 and TIFA.
(i) Rad53: The budding yeast homolog of Checkpoint kinase 2. Rad53 controls the S-phase checkpoint as well as the G1 and G2 DNA damage checkpoints. The kinase activity of Rad53 is dependent on its autophosphorylation, which is supported by dimerization. The open and closed intermediate conformations of Rad53 kinase domain were revealed through X-ray crystallography using Rad532-466 (consist of SCD1, FHA1 and Kinase domain: Rad53SFK). The open and closed forms of Rad53SFK differ in the relative rotation and movement of αC helix in the N-lobe. In the open form, the αC helix is further away from the β3 strand. In the closed form, the αC helix is closer to the β3 strand and the E244 on the αC helix forms the conserved salt bridge with K227 on the β3 strand, which has been shown to be crucial for the stable binding with ATP. In addition, the SAXS analyses of Rad53SFK and phospho-Rad53SFK showed that the Rad53SFK has various conformations in solution while the FHA domains and kinase domain of phospho-Rad53SFK are in close proximity in solution. The results provided important insights into how the autophosphorylation mechanism of Rad53 is regulated by phospho-priming induced dimerization, and why proximity driven by dimerization is distinct from that induced by concentration.
(ii) TIFA: TRAF-interacting protein with a FHA domain. TIFA is involved in the activation of NF-κB. Upon stimulation, TIFA is phosphorylated at Thr9, which triggers TIFA oligomerization. The oligomeric form of TIFA could promote TRAF6 oligomerization and ubiquitination, which activates NF-κB. The structural information of TIFA (tTIFA1-150) and its complex with TIFA N-terminal phosphorylated Thr9 peptide 1-15 (pThr9-TIFA1-15) showed the unique structure and the novel binding mode of TIFA FHA domain. The intrinsic TIFA dimer has a distinct dimer interface, and its structural features revealed that pThr9-FHA domain binding is between inter-dimers rather than intra-dimers. The structural information helped us gained insight into the TIFA dimer, and uncovered the molecular mechanism for the functionally important oligomerization of TIFA.

並列關鍵字

FHA domian ； TIFA ； Rad53

參考文獻

1. Hofmann, K. and P. Bucher, The FHA domain: a putative nuclear signalling domain found in protein kinases and transcription factors. Trends Biochem Sci, 1995. 20(9): p. 347-9.

2. Liao, H., I.J. Byeon, and M.D. Tsai, Structure and function of a new phosphopeptide-binding domain containing the FHA2 of Rad53. J Mol Biol, 1999. 294(4): p. 1041-9.

3. Durocher, D., et al., The molecular basis of FHA domain:phosphopeptide binding specificity and implications for phospho-dependent signaling mechanisms. Mol Cell, 2000. 6(5): p. 1169-82.

4. Durocher, D., et al., The FHA domain is a modular phosphopeptide recognition motif. Mol Cell, 1999. 4(3): p. 387-94.

5. Liao, H., et al., Structure of the FHA1 domain of yeast Rad53 and identification of binding sites for both FHA1 and its target protein Rad9. J Mol Biol, 2000. 304(5): p. 941-51.