利用核磁共振探討人類鈣離子結合S100A11蛋白質水溶液結構以及其與RAGE V-domain之間的交互作用

NMR Structure of Calcium Bound Human S100A11 and Its Interaction with RAGE V-Domain

張原銘(Yuan-Ming Chang)

指導教授 ： 余靖

國立清華大學/理學院/化學系/碩士(2012年)

摘要

人類S100A11為一雙聚體蛋白質，其具有可以與鈣離子結合的EF-hand，而與鈣離子結合後會改變其構形並與目標蛋白交互作用，而RAGE為其中一種會與S100A11結合的目標蛋白。RAGE隸屬於表面細胞分子中的免疫球蛋白，與不同的配體結合後，可以誘導不同的細胞訊息傳遞，因此在許多疾病當中扮演非常重要的角色。
在第二章中，我們利用疏水性管柱以及鈷離子親和性管柱分別純化人類S100A11蛋白質與RAGE V蛋白質，而在第三、四章中，由於人類S100A11與鈣離子結合的結構目前尚未清楚，因此我們希望藉由NMR解出其水溶液結構，並進一步探討其與配體之間的交互作用。我們使用多維度核磁共振實驗如HNCA、HN(CO)CA、HCCH-TOCSY以及HCCH-COSY等來完成所有的化學位移判定，並利用實驗所得的距離、雙面角、氫鍵限制條件以及ARIA/CNS軟體解出人類鈣離子結合S100A11水溶液結構。
在第五章中，為了瞭解S100A11與RAGE分子層級的結合區域，我們利用二維1H-15N HSQC滴定實驗來找出S100A11與RAGE可能的結合位置，並且利用螢光實驗以及分析級超高速離心機探討他們之間的交互作用，從這些數據中，我們發現S100A11與RAGE確實有形成複合物的可能性存在。

關鍵字

S100A11蛋白質 ； RAGE蛋白質 ； 核磁共振

並列摘要

Human S100A11 is a homodimeric EF-hand calcium binding protein that undergoes a calcium-induced conformational change and interacts with target protein. RAGE is a multiligand receptor binding to S100A11 and the interactions at molecular level have not been reported. It is a member of the immunoglobulin protein family of cell surface molecules. RAGE plays an important role in many human pathologies by inducing cellular signaling actions upon binding of different ligands.
In chapter 2, we purified human S100A11 and RAGE V by using phenyl-sepharose column and cobalt affinity column. In chapter 3 and 4, chemical shift assignments were made from HNCA, HN(CO)CA, HCCH-TOCST and HCCH-COSY,etc. Furthermore, the solution structure of human calcium bound S100A11 was determined by ARIA/CNS software using the experimental restraints such as distance, dihedral angle, hydrogen bond.
In chapter 5, we used a variety of biophysical methods, including 2D 1H-15N HSQC titration, fluorescence spectroscopy and analytical ultracentrifugation, to characterize the binding interface regions and interactions between S100A11 and RAGE at the molecular level. Finally, we postulate that S100A11 and RAGE might interact with each other.

參考文獻

[21] B. I. Hudson, A. Z. Kalea, M. del Mar Arriero, E. Harja, E. Boulanger, V. D'Agati, A. M. Schmidt, Journal of Biological Chemistry 2008, 283, 34457-34468.

[24] M. A. Hofmann, S. Drury, C. Fu, W. Qu, A. Taguchi, Y. Lu, C. Avila, N. Kambham, A. Bierhaus, P. Nawroth, M. F. Neurath, T. Slattery, D. Beach, J. McClary, M. Nagashima, J. Morser, D. Stern, A. M. Schmidt, Cell 1999, 97, 889-901.

[19] K. Nakamura, S.-i. Yamagishi, Y. Nakamura, K. Takenaka, T. Matsui, Y. Jinnouchi, T. Imaizumi, Microvascular Research 2005, 70, 137-141.

[12] aA. A. Memon, B. S. Sorensen, P. Meldgaard, L. Fokdal, T. Thykjaer, E. Nexo, Clinical Cancer Research 2005, 11, 606-611; bE. Leclerc, G. Fritz, S. W. Vetter, C. W. Heizmann, Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 2009, 1793, 993-1007.

[28] N. M. Marlatt, D. E. Spratt, G. S. Shaw, Protein Expression and Purification 2010, 73, 58-64.