FAK衍生磷酸化胜肽和Grb2-SH2的結合模式之晶體結構探討

Crystal structure of the Grb2-SH2 domain in complex with a FAK-derived phosphotyrosyl peptide

陳孝信(Hsiao-Hsin Chen)

指導教授 ： 段葉芳

國立臺北科技大學/工程學院/生物科技研究所/碩士(2010年)

摘要

Focal adhesion kinase (FAK)是一個細胞內的酪胺酸磷酸酶，它被活化後會產生一連串細胞的生理反應，包括細胞的吸附、遷移、入侵、增生和存活。在一些癌症的病例中，常會見到FAK有過量的表現，因此FAK有潛力作為一個抗癌的藥物標的。FAK上酪胺酸的磷酸化對訊息傳遞的調控扮演重要的角色，其中一個在FAK上第925個胺基酸酪胺酸(Tyrosine)，位於螺旋1上，存在有Grb2-SH2 domain的結合序列pYXNX，當Y925被磷酸化後，就會與Grb2-SH2結合，接著活化Ras-MAPK的訊息傳遞路徑。多數的SH2 domain會和延伸的胜肽結合，但Grb2-SH2內Trp的側鏈會防止它和延伸的胜肽形成β-turn構形的胜肽結合，然而FAK-FAT domain上的Y925卻位於α螺旋的結構中。本研究想了解的重點是，是否FAT上的螺旋1在與Grb2-SH2結合後產生構型的變化？如此便能更清楚此兩個重要分子的結合模式及調控情形，未來或能設計抑制劑阻斷此一結合。本研究將pY925/Grb2-SH2的複合物進行晶體的篩選，結果在一條件下有單晶的形成，利用同步輻射的光源收集晶體的繞射數據，收到一解析度為2.49A的繞射光譜，並且利用分子取代法成功解決相位角的問題，得到複合物的結構我們得到的結晶結構其在一個不對稱單元中包含有六個Grb2-SH2分子、兩個磷酸化分子、六個磷酸離子、二十四個水分子。此結構中我們發現，Trp121在有磷酸化的胜肽和沒有磷酸化胜肽結合的會有不同的構型，且兩種構型同時存在於同一個晶體結構中，Grb2-SH2上的BC loop會參與Grb2-SH2和磷酸化胜肽的結合。過去關於Grb2-SH2結構探討的資料，Grb2-SH2 only以及Grb2 complex都是在不同晶體中得到，但是我們的結果和前人的最大差別是，我們在同一個晶體中同時得到了Grb2-SH2 only以及Grb2-SH2 complex的結構，這個結果方便我們探討去除掉不同養晶環境而造成結構上的差異，使結果能表現出如同在同一環境下的情況。

關鍵字

同步輻射 ； 蛋白質結晶 ； 生長因子接受蛋白2

並列摘要

The focal adhesion targeting (FAT) domain contained tyrosine 925 (Y925) of focal adhesion kinase (FAK) is critical for recruitment of FAK to focal adhesions. FAK mediates signaling through Grb2-SH2 domain via phosphorylated Y925. Previous studies have shown that FAT is a four-helix bundle structure and Y925 is located in the first helix of the domain. However, the typical conformation of phosphopeptide bound to Grb2-SH2 domain is β-turn structure. It makes a huge controversy over the possible adaptation of pY925 on the Grb2-SH2 domain bound form. Hence, the structural determination of Grb2-SH2 domain complex with a phosphotyrosine(pY925)-peptide derived from FAK was carried out by X-ray diffraction method. Diffraction data were collected to 2.49 A resolution on BL13C1 beamline of NSRRC from a flash-frozen crystal at 100 K. The crystals belonged to space group P3121, with unit-cell parameters a=102.656, b=102.656, c=127.611 A. Six Grb2-SH2 domain molecules were comprised in an asymmetric unit, but there were only two molecules complexed with phosphopeptide. In addition, the presence of Trp121 residue prevents the linear recognition of phosphopeptide at the surface of the Grb2-SH2 domain, resulting in a bent conformation for the FAK peptide NDKV(pY)ENVTG. Although the electron density map of N- and C-termini of peptide were missed, the central residues KV(pY)ENV of peptide exists the unambiguous map for the elucidation of molecular recognition. The structural information of the Grb2-SH2 and FAK peptide complex may not resulting in understanding of the communication between signaling molecules, but also shed light on the design of new inhibitors with cancer therapeutic application.

並列關鍵字

Grb2 ； SH2 domain ； FAK ； synchrotron radiation ； crystallization.

參考文獻

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