果蠅中酪氨酸亞硫酸基轉移酶與胜肽交互作用之探討

Characterization of Protein-peptide Interactions of Tyrosylprotein Sulfotransferase (TPST) from Drosophila melanogaster

辛塔(Shinta Anggia Murni)

指導教授 ： 陳俊榮

國立清華大學/理學院/物理學系/碩士(2021年)

摘要

蛋白質酪氨酸的硫酸化作用是透過細胞膜上酪氨酸亞硫酸基轉移酶 (TPST) 所催化，而酪氨酸亞硫酸基轉移酶具有3'-磷酸腺苷-5'-磷酸硫酸酯 (PAPS) 作為輔助因子進行的目標蛋白的後修飾。要硫酸化蛋白質，需透過PAPS合成酶活化3'-磷酸腺苷-5'-磷酸硫酸鹽 (PAPS) 的硫酸鹽，再將3'-磷酸腺苷-5'-磷酸硫酸鹽 (PAPS) 中的硫基轉移到特定的目標蛋白質和胜肽中的酪氨酸殘基上。除阿拉伯芥 (Arabidopsis) 和果蠅 (Drosophila melanogaster) 僅具有一種TPST之外，其他所有生物均具有兩種TPST。果蠅TPST，稱為DmTPST (果蠅酪氨酸亞硫酸基轉移酶)。
DmTPST蛋白質從大腸桿菌中的BL21-CodonPlus（DE3）-RIL細胞中萃取，其中PET21b為載體。總序列共306氨基酸，其等電位點之理論值為6.73，分子量為37 kDa。本實驗所使用的DmTPST是從Trp115突變為Ala115，稱為DmTPST-W115A，而突變的目的是為了獲得的較均一性的單體蛋白，以有利進行蛋白質晶體的培養。在結晶之前需要針對DmTPST-W115A進行純化。純化第一步驟是使用NiSO4管柱進行親和層析，以親和性大小從其他蛋白分子中分離出目標蛋白。 第二步驟是使用EnrichTM SEC 650 nm管柱進行凝膠過濾層析，以分子質量分離目標蛋白，從而使純化的蛋白達到高純度。
之後，利用在蒸氣擴散結晶方法篩選與改良多種長晶條件，但所獲得的晶體品質不佳，無法成功取得X光繞射數據。因此，我們使用了對接軟體HADDOCK對DmTPST-W115A蛋白與PSGL-1胜肽之間的相互作用進行了模型建立，而在計算上我們採用了TPST含有PAP和不含PAP的兩種模式。結果顯示，在含有PAP的蛋白質-胜肽相互作用比沒有PAP的蛋白質具有更好的功能解釋，因為PSGL-1胜肽結合蛋白與位置接近DmTPST的催化區域及PAP，而PSGL-1胜肽的相對位置與結合模式也與之前文獻中人類TPST上 的C4胜肽相似，這意含本研究的果蠅TPST結果可以用來闡明人類TPST的催化機制。這種交互作用機制從根本上增強了複合物結構的穩定性，維持了蛋白質的折疊，並大幅度地減少了生物分子中水界面的相互作用。由分析結果顯示PSGL-1上的兩個主要酪氨酸殘基是Tys607和Tys610，它們可被DmTPST-W115A辨識，從而證明了蛋白質界面殘基 (Arg101 、Arg105、Thr200、Gln108和His112) 與受質胜肽之間的許多結合的作用，例如氫鍵，疏水鍵，鹽橋和π-陽離子等相互作用。

關鍵字

no ； 果蠅中酪 ； 氨酸亞硫酸基 ； 酶與胜肽交互作用 ； 中酪氨酸亞硫酸基 ； 酶與胜肽

並列摘要

Protein tyrosine sulfation is catalyzed by membrane tyrosylprotein sulfotransferase (TPST) with 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as a co-factor, which is a process of a post-translasional modification for the target protein. The process involves the actived sulfate of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) through PAPS synthetase to transfer the sulfur group from 3'- phosphoadenosine-5'-phosphosulfate (PAPS) into a specific tyrosine residue of the target proteins and peptides, which is needed for their functions. Almost all of the organisms have two kinds of TPST except Arabidopsis and Drosophila melanogaster that have only one form.
TPST from Drosophila melanogaster (fruit fly), called DmTPST, was extracted from BL21-CodonPlus (DE3)-RIL competent cells in Escherichia coli bacteria from the mutant W115A of DmTPST with PET21b as the palsmid. There are total 306 amino-acid residues with a theoretical isoelectric point 6.73 and a molecular mass 37 kDa. DmTPST, called DmTPST-W115A, has been mutated from Try115 to Ala115 to obtain the pure monomeric protein of DmTPST, presumably making a higher possibility of crystallization. The purification is needed in advance of the crystallization. The first purification was the affinity-chromatography with a NiSO4 column to isolate the target protein from other proteins. The second step was the gel filtration (size-exclusion chromatography) using an EnrichTM SEC 650 nm column to isolate the target protein by molecule masses to obtain the purified protein of a good quality.
Furthermore, the crystallization of the purified protein was first screened with the vapor-diffusion method with the various conditions. However, the crystallization of DmTPST was not successful despite a number of attempts. Alternatively, we used the docking software HADDOCK to model the complex structure to study the interactions between the DmTPST-W115A protein and the PSGL-1 peptide, with and without PAP in TPST, respectively. The protein-peptide interactions with PAP has the better reliability than that without PAP because the binding geometry and location PSGL-1 peptide are demonstrated to be close to the catalytic site of TPST, which is similar to a C4 peptide at the human TPST protein. This mechanism fundamentally enhances the stability of complex structure, maintans the protein folding and minimizes the water interface interaction in biological system of molecules. The two main tyrosine residues on PSGL-1 are Tys607 and Tys610, which are recognized by DmTPST-W115A, demonstrate several interactions between the substrate ligand and the interface residues (Arg101, Arg105, Thr200, Gln108, and His112) of protein through the hydrogen bond, hydrophobic bond, salt bridge, and π-cation interactions.

並列關鍵字

no ； Tyrosylprotein Sulfotransferase ； Protein-peptide Interactions ； Drosophila melanogaster ； PAP ； HADDOCK

參考文獻

References

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