利用核磁共振技術探討人類鈣離子結合之S100B在S100B-SIP189-219複合物內的水溶液結構以及其與SIP189-219蛋白之間的交互作用

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研究生：	謝育程 Xie, Yu-Cheng
論文名稱：	利用核磁共振技術探討人類鈣離子結合之S100B在S100B-SIP189-219複合物內的水溶液結構以及其與SIP189-219蛋白之間的交互作用 NMR Solution Structure of Calcium-bound Human S100B in Complexed with SIP189-219 and its Interation with SIP189-219
指導教授：	余靖
口試委員:	陳金榜莊偉哲
學位類別：	碩士 Master
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2013
畢業學年度：	101
語文別：	中文
論文頁數：	101
中文關鍵詞：	核磁共振、解離常數、螢光、化學位移
相關次數：	點閱：2 下載：0
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人類S100B隸屬於S100蛋白質家族，在人體中具有多樣的生理活性。其具有EF-hand之結構，能與鈣離子結合而改變構形並與多種蛋白質作用。在細胞內SIP與Siah-1、Skp-1、Ebi ...等蛋白協同參與並調節β-catenin的含量，間接影響Tcf/LEF轉錄因子的活性。有報導指出S100蛋白會與SIP中的SGS domain結合而抑制β-catenin的水解，導致癌細胞的增生。
在本文我們希望計算出在複合物中S100B 之水溶液結構，並研究其與SIP之間的交互作用。我們利用一系列的三維核磁共振實驗完成其骨架和支鏈的化學位移判定，再利用ARIA/CNS軟體解出複合物中S100B之水溶液結構。
接著使用HSQC滴定的實驗觀測在交互作用中受到最多化學位移擾動的氨基酸，搭配我們計算出的複合物中S100B結構和已發表存在S100A6複合物中的SIP189-219結構，並以此作為運行HADDOCK的資訊。結果顯示S100B- SIP189-219複合物的結構比S100A6- SIP189-219複合物的結構較為外擴。此外，我們也用恆溫滴定微卡計與螢光技術求得其解離常數，以證實S100B與SIP189-219有交互作用存在。

S100B, a member of S100 family, contains various biological activities in the human body. There are two EF-hand motifs, which may interact with calcium ions, in the protein. Upon calcium binding, S100B undergoes conformational changes to expose a hydrophobic surface as a binding site for various target proteins. For example, the target proteins such as Siah-1, Skp-1 and Ebi associate with Ca2+-S100B and subsequently adjust the expression level of β-catenin and the transcription activity of Tcf/LEF. Several studies indicate that S100 proteins may interact with the SGS domain of SIP to inhibit β-catenin degradation and enhance the proliferation of certain cancer cells.
In this thesis, we determine the solution structure of Ca2+-S100B in complex and study its interaction with SIP using ITC and fluorescence spectroscopy. By using a series of 3D NMR experiments, the backbone and side chain resonaces were alomost completely assigned.
HSQC titration experiments indicate the residues on one protein with significant chemical shift perturbation after associating with its binding partner. The chemical shift perturbation data on both S100B and SIP proteins serve as the input constraints for HADDOCK calculation. The atomic coordinates of S100B in complex calculated in the study were docked with the published structure of SIP189-219 bound to S100A6. The result reveals that the complex structure of S100B-SIP189-219 is more expanded then that of S100B-SIP189-219.

謝誌 ........................................................................................................................................... II
Abstract .................................................................................................................................... III
摘要 .......................................................................................................................................... IV
圖目錄 ...................................................................................................................................... IV
縮寫表 ...................................................................................................................................... VI
第一章 前言 ........................................................................................................................... 1
1-1 S100B 之性質與結構 ................................................................................................. 1
1-2 CacyBP/SIP之性質與結構 ......................................................................................... 3
1-3 S100蛋白與SIP的交互作用 ..................................................................................... 5
1-4 實驗動機 ...................................................................................................................... 6
1-5 生物核磁共振技術介紹 .............................................................................................. 7
1-6 蛋白質分子的NMR光譜循序判定(sequential assignment) ..................................... 8
1-6.1 蛋白質分子的骨架循序判定 ............................................................................... 8
1-6.2 蛋白質分子的支鏈循序判定 ............................................................................. 10
1-7 限制條件的找尋 ........................................................................................................ 11
1-7.1 距離限制條件(NOE distance constraints) .......................................................... 11
1-7.2 雙面角限制條件 ................................................................................................. 11
1-7.3 氫鍵限制條件 ..................................................................................................... 12
1-7.4 結構計算 ............................................................................................................. 12
1.8 HADDOCK(high ambiguity driven docking)介紹 .................................................... 13
第二章 材料與方法 ............................................................................................................... 16
2-1 蛋白質的表現與純化 .................................................................................................. 16
2-1.1 人類S100B蛋白質之取得 .................................................................................. 16
2-1.2人類S100B蛋白質之表現 ................................................................................... 16
2-1.3 人類S100B蛋白質之純化 .................................................................................. 18
II
2-1.4 SIP(殘基189-219)蛋白之取得與表現 .............................................................. 21
2-1.5人類SIP189-219蛋白質之表現 ................................................................................ 21
2-1.6 SIP189-219蛋白之純化 .......................................................................................... 23
2-1.7蛋白質濃度的測定 ................................................................................................ 26
2-2 蛋白質之基本性質鑑定 .............................................................................................. 27
2-2.1蛋白質質量鑑定 .................................................................................................... 27
2-3 蛋白質之間的交互作用 .............................................................................................. 29
2-3.1 螢光放射光譜(Fluorescence spectrum) ............................................................... 30
2-3.2恆溫滴定微卡計(isothermal titration calorimetry, ITC) ....................................... 31
2-3.3 核磁共振實驗 ....................................................................................................... 33
第三章 結果與討論 ............................................................................................................... 38
3-1 S100B與SIP189-219蛋白質在大腸桿菌之表現 ........................................................ 38
3-1.1 異丙基硫化半乳糖(IPTG)對大腸桿菌之誘導作用 ........................................... 38
3-1.2 S100B與SIP 189-219蛋白質的大量表現 ............................................................ 39
3-2. S100B蛋白之純化 ...................................................................................................... 41
3-2.1 陰離子交換層析管柱(monoQ)純化 .................................................................... 41
3-2.2 疏水交互作用層析管柱(HIC) 純化 ................................................................... 42
3-3 SIP 189-219融合蛋白之純化 ........................................................................................... 43
3-3.1鈷離子親和性管柱純化 ........................................................................................ 43
3-3.2 SIP189-219蛋白與Trx tag之分離 ........................................................................ 44
3-5 S100B及與SIP 189-219蛋白質之分子量鑑定 ........................................................... 45
3-6 S100B與SIP 189-219蛋白質之二級結構鑑定 ........................................................... 47
3-7 在複合物中(S100B與SIP 189-219複合物)S100B之NMR實驗 ............................... 48
3-7.1在複合物中S100B蛋白之1H-15N HSQC ........................................................... 49
3-7.2在複合物中S100B蛋白之HNCA與HN(CO)CA ............................................. 50
3-7.3在複合物中S100B蛋白之CACB(CO)NH ......................................................... 51
III
3-7.4在複合物中S100B蛋白之HNCO ....................................................................... 52
3-7.5在複合物中S100B蛋白之CC(CO)NH ............................................................... 53
3-7.6在複合物中S100B蛋白之HBHA(CO)NH ......................................................... 54
3-7.7在複合物中S100B蛋白之TOCSY和 COSY ................................................... 55
3-7.8在複合物中S100B蛋白之15N-edited 和13C-edited NOESY ....................... 55
3-8 在複合物中S100B之二級結構預測 ......................................................................... 56
3-8.1 TALOS ................................................................................................................ 56
3-8.2 CSI(Chemical shift index) ..................................................................................... 58
3-9 在複合物中S100B蛋白質結構計算 ......................................................................... 59
3-10 在複合物中S100B結構計算與已發表的未結合S100B ....................................... 63
3-11 在複合物中SIP189-219 NMR實驗 .............................................................................. 63
3-12 S100B與SIP189-219交互作用之探討 ...................................................................... 65
3-12.1 S100B(M9)與SIP189-219 的HSQC滴定實驗 ................................................... 65
3-12.2 SIP189-219 (M9)與S100B的HSQC滴定實驗 .................................................. 66
3-12.3 S100B的結合位置 ............................................................................................ 68
3-12.4 HADDOCK(High Ambiguity Driven Docking)................................................. 69
3-12.5 ITC .................................................................................................................... 71
3-12.6 螢光 ................................................................................................................... 72
第四章 結論 ........................................................................................................................... 76
參考文獻 ................................................................................................................................. 77
附錄一：在複合物中S100B的HNCA和HN(CO)CA的strips ........................................ 80
附錄二：在複合物中SIP189-219的HNCA和HN(CO)CA的strips ..................................... 83
附錄三：在複合物中S100B完整化學位移表 .................................................................... 85
                                

參考文獻
1. Marenholz I, Heizmann CW, Fritz G. S100 proteins in mouse and man: from evolution to function and pathology (including an update of the nomenclature). Biochemical and Biophysical Research Communications 2004; 322: 1111-1122.
2. Marenholz I, Lovering RC, Heizmann CW. An update of the S100 nomenclature. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 2006; 1763: 1282-1283.
3. A. Messerschmidt WB, &M. Cygler, eds. Hanbool of Metalloproteins. 2004; 3: 529-540.
4. Bhattacharya S, Bunick C, Chazin W. Target selectivity in EF-hand calcium binding proteins. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 2004; 1742: 69-79.
5. Vallely KM, Rustandi RR, Ellis KC et al. Solution Structure of Human Mts1 (S100A4) As Determined by NMR Spectroscopy†. Biochemistry 2002; 41: 12670-12680.
6. Ikura M, Ames JB. Genetic polymorphism and protein conformational plasticity in the calmodulin superfamily: Two ways to promote multifunctionality. Proceedings of the National Academy of Sciences of the United States of America 2006; 103: 1159-1164.
7. Wright NT, Cannon BR, Wilder PT et al. Solution Structure of S100A1 Bound to the CapZ Peptide (TRTK12). Journal of Molecular Biology 2009; 386: 1265-1277.
8. Donato R. S100: a multigenic family of calcium-modulated proteins of the EF-hand type with intracellular and extracellular functional roles. The International Journal of Biochemistry & Cell Biology 2001; 33: 637-668.
9. Van Eldik LJ, Zimmer DB. Secretion of S-100 from rat C6 glioma cells. Brain Research 1987; 436: 367-370.
10. Pinto SS, Gottfried C, Mendez A et al. Immunocontent and secretion of S100B in astrocyte cultures from different brain regions in relation to morphology. FEBS Letters 2000; 486: 203-207.
11. Steiner J, Bernstein HG, Bogerts B et al. S100B is expressed in, and released from, OLN-93 oligodendrocytes: Influence of serum and glucose deprivation. Neuroscience 2008; 154: 496-503.
12. Perrone L, Peluso G, Melone MAB. RAGE recycles at the plasma membrane in S100B secretory vesicles and promotes Schwann cells morphological changes.78 Journal of Cellular Physiology 2008; 217: 60-71.
13. Baudier J, Glasser N, Gerard D. Ions binding to S100 proteins. I. Calcium- and zinc-binding properties of bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) protein: Zn2+ regulates Ca2+ binding on S100b protein. Journal of Biological Chemistry 1986; 261: 8192-8203.
14. Nishikawa T, Lee ISM, Shiraishi N et al. Identification of S100b Protein as Copper-binding Protein and Its Suppression of Copper-induced Cell Damage. Journal of Biological Chemistry 1997; 272: 23037-23041.
15. Drohat AC, Amburgey JC, Abildgaard F et al. Solution Structure of Rat Apo-S100B(ββ) As Determined by NMR Spectroscopy†,‡. Biochemistry 1996; 35: 11577-11588.
16. Kilby PM, Van Eldik LJ, Roberts GCK. The solution structure of the bovine S100B protein dimer in the calcium-free state. Structure 1996; 4: 1041-1052.
17. Drohat AC, Baldisseri DM, Rustandi RR, Weber DJ. Solution Structure of Calcium-Bound Rat S100B(ββ) As Determined by Nuclear Magnetic Resonance Spectroscopy†,‡. Biochemistry 1998; 37: 2729-2740.
18. Matsumura H, Shiba T, Inoue T et al. A novel mode of target recognition suggested by the 2.0 å structure of holo S100B from bovine brain. Structure (London, England : 1993) 1998; 6: 233-241.
19. Smith SP, Shaw GS. A novel calcium-sensitive switch revealed by the structure of human S100B in the calcium-bound form. Structure 1998; 6: 211-222.
20. Rust RR, Baldisseri DM, Weber DJ. Structure of the negative regulatory domain of p53 bound to S100B([beta][beta]). Nat Struct Mol Biol 2000; 7: 570-574.
21. Bhattacharya S, Large E, Heizmann CW et al. Structure of the Ca2+/S100B/NDR Kinase Peptide Complex: Insights into S100 Target Specificity and Activation of the Kinase†. Biochemistry 2003; 42: 14416-14426.
22. Inman KG, Yang R, Rustandi RR et al. Solution NMR Structure of S100B Bound to the High-affinity Target Peptide TRTK-12. Journal of Molecular Biology 2002; 324: 1003-1014.
23. Hartman KG, Wilder PT, Varney K et al. Inhibiting S100B in Malignant Melanoma. 2013.
24. Krebs J, Heizmann CW. Calcium-binding proteins and the EF-hand principle. In Joachim K, Marek M (eds): New Comprehensive Biochemistry. Elsevier 2007; 51-93.
25. Matsuzawa S-i, Reed JC. Siah-1, SIP, and Ebi Collaborate in a Novel Pathway for β-Catenin Degradation Linked to p53 Responses. Molecular Cell 2001; 7: 915-926.
26. Hershko A, Ciechanover A. THE UBIQUITIN SYSTEM. Annual Review of 79 Biochemistry 1998; 67: 425-479.
27. Mann B, Gelos M, Siedow A et al. Target genes of β-catenin–T cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas. Proceedings of the National Academy of Sciences 1999; 96: 1603-1608.
28. Morin PJ. β-catenin signaling and cancer. BioEssays 1999; 21: 1021-1030.
29. Bhattacharya S, Lee Y-T, Michowski W et al. The Modular Structure of SIP Facilitates Its Role in Stabilizing Multiprotein Assemblies†,‡. Biochemistry 2005; 44: 9462-9471.
30. Santelli E, Leone M, Li C et al. Structural Analysis of Siah1-Siah-interacting Protein Interactions and Insights into the Assembly of an E3 Ligase Multiprotein Complex. Journal of Biological Chemistry 2005; 280: 34278-34287.
31. Kitagawa K, Skowyra D, Elledge SJ et al. SGT1 Encodes an Essential Component of the Yeast Kinetochore Assembly Pathway and a Novel Subunit of the SCF Ubiquitin Ligase Complex. Molecular Cell 1999; 4: 21-33.
32. Azevedo C, Sadanandom A, Kitagawa K et al. The RAR1 Interactor SGT1, an Essential Component of R Gene-Triggered Disease Resistance. Science 2002; 295: 2073-2076.
33. Filipek A, Wojda U. p30, a novel protein target of mouse calcyclin (S100A6). Biochem. J. 1996; 320: 585-587.
34. Filipek A, Kuźnicki J. Molecular Cloning and Expression of a Mouse Brain cDNA Encoding a Novel Protein Target of Calcyclin. Journal of Neurochemistry 1998; 70: 1793-1798.
35. Filipek A, Jastrzebska B, Nowotny M, Kuznicki J. CacyBP/SIP, a Calcyclin and Siah-1-interacting Protein, Binds EF-hand Proteins of the S100 Family. Journal of Biological Chemistry 2002; 277: 28848-28852.
36. Lee Y-T, Dimitrova YN, Schneider G et al. Structure of the S100A6 Complex with a Fragment from the C-Terminal Domain of Siah-1 Interacting Protein: A Novel Mode for S100 Protein Target Recognition†‡. Biochemistry 2008; 47: 10921-10932.
37. Xiaoxuan N, Shiren S, Kun Z et al. S100A6 Protein Negatively Regulates CacyBP/SIP-Mediated Inhibition of Gastric Cancer Cell Proliferation and Tumorigenesis. PLoS ONE 2012; 7.
38. Dominguez C, Boelens R, Bonvin AMJJ. HADDOCK: A Protein−Protein Docking Approach Based on Biochemical or Biophysical Information. Journal of the American Chemical Society 2003; 125: 1731-1737.
39. Smith S, Shaw G. Assignment and secondary structure of calcium-bound human S100B. Journal of Biomolecular NMR 1997; 10: 77-88.

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