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

謝育程(Yu-Cheng Xie)

指導教授 ： 余靖

國立清華大學/理學院/化學系/碩士(2013年)

摘要

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。

關鍵字

核磁共振 ； 解離常數 ； 螢光 ； 化學位移

並列摘要

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.

參考文獻

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