(1)阿拉伯芥葉綠體外膜轉位子Toc33的結構功能研究Structural and functional study of chloroplast translocon component atToc33 (2)台灣眼鏡蛇毒磷脂質水解酶A2與脂肪酸複合體的結晶學研究Crystallographic study of cobra phospholipase A2 complexed with fatty acid

(1)Structural and functional study of chloroplast translocon component atToc33; (2)Crystallographic study of cobra phospholipase A2 complexed with fatty acid

葉義弘(Yi-Hung Yeh)

指導教授 ： 孫玉珠 ； 蕭傳鐙

國立清華大學/生命科學院/生命科學系/博士(2008年)

https://doi.org/10.6843/NTHU.2008.00534

摘要

(一)阿拉伯芥葉綠體轉位子Toc33的結構功能研究

摘要
阿拉伯芥葉綠體轉位子Toc33是一個三磷酸鳥苷酸水解酶(GTPase)，而且是幫助前軀蛋白質運送到葉綠體內的葉綠體外膜轉位機組(Toc)複合體的一員。依據psToc34結晶結構的研究，Arg130(對應於psToc34的Arg133)，是與atToc33雙體形成有關，而且藉由此雙體來進行分子間的三磷酸鳥苷酸水解酶活化作用。這裡我們發表解析度3.2Ǻ的atToc33突變株, atToc33(R130A)的結晶結構。在水溶液及晶體中，atToc33(R130A)都是呈現的是單體的構形。相形之下，在atToc33原生株及豌豆Toc三磷酸鳥苷酸水解酶同源蛋白，psToc159，兩者都可以在水溶液中形成雙體。與單體的atToc33，atToc159和atToc33(R130A)相比，雙體的atToc33及psToc159都呈現顯著且較高的三磷酸鳥苷酸水解酶活性。根據本論文研究結果指出，Arg130是雙體形成的關鍵因素，而且Arg130對其三磷酸鳥苷酸水解酶的活性也很重要。所以藉由雙體形成的三磷酸鳥苷酸水解酶活性作用似乎是蛋白質進入葉綠體內的一個關鍵調節步驟。

(二)台灣眼鏡蛇毒磷脂質水解酶A2與脂肪酸複合體的結晶學研究

摘要
磷脂質水解酶經由介面活化作用可在受質凝集狀態表現出高催化活性。由於近來PLA2結合在細胞膜表面的生物物理特性已被認為是帶負電的雙極性分子協同結合到細胞膜介面和其具調節性的N端helix有關，兩者也都與造成介面活化作用相關。所以我們發表台灣眼鏡蛇毒PLA2與帶負電的硫酸根雙極性分子結合在介面及催化部位的結構，也利用傅立葉轉換紅外光光譜(FTIR)決定了PLA2與磷脂質結合的位向。研究結果顯示PLA2與磷脂質細胞膜結合時會隨著此酶的芳香環殘基穿透膜造成參差不齊的深度促使磷脂質的疏水性端彎曲翹起。此介面的雙極性分子會經由構形改變，從介面結合部位往活化部位擴散，並印證在隨著時間會改變晶體的形狀上。因此，台灣眼鏡蛇毒PLA2的介面活化作用可能意味是此酶在介面與雙極性分子結合所產生的結構中間態會幫助受質擴散至活化區。

關鍵字

葉綠體 ； 三磷酸鳥苷酸水解酶 ； 葉綠體外膜轉位機組 ； 雙體化作用 ； 結晶結構 ； 三磷酸鳥苷酸活化蛋白 ； 磷脂質水解酶A2 ； 傅立葉轉換紅外光光譜 ； 介面結合 ； 雙極性分子結合 ； HASH(0x1c583c40)

並列摘要

(1)Structural and functional study of chloroplast translocon component atToc33

ABSTRACT
Arabidopsis Toc33 (atToc33) is a GTPase and a member of the Toc (translocon at the outer-envelope membrane of chloroplasts) complex that associates with precursor proteins during protein import into chloroplasts. By inference from the crystal structure of psToc34, a homologue in pea, the arginine at residue 130 (Arg130) has been implicated in formation of the atToc33 dimer and inter-molecular GTPase activation within the dimer. Here we report the crystal structure at 3.2 Å resolution of an atToc33 mutant, atToc33(R130A), in which Arg130 was mutated to alanine. Both in solution and in crystals, atToc33(R130A) was present in its monomeric form. In contrast, both wild-type atToc33 and another pea Toc GTPase homologue, pea Toc159 (psToc159), were able to form dimers in solution. Dimeric atToc33 and psToc159 had significantly higher GTPase activity than monomeric atToc33, psToc159 and atToc33(R130A). Molecular modeling using the structures of psToc34 and atToc33(R130A) suggests that, in an architectural dimer of atToc33, Arg130 from one monomer interacts with the □-phosphate of GDP and several other amino acids of the other monomer. These results indicate that Arg130 is critical for dimer formation, which is itself important for GTPase activity. Activation of GTPase activity by dimer formation is likely to be a critical regulatory step in protein import into chloroplasts.

(2)Crystallographic study of cobra phospholipase A2 complexed with fatty acid

ABSTRACT
Phospholipase A2 (PLA2) exhibit high catalytic activities on aggregated substrates via interfacial activation. Recent biophysical characterizations of PLA2 bound to a membrane surface have suggested that both cooperative binding of anionic amphiphiles to the interface and conformational changes of the regulatory N-terminal helix are involved in interfacial activation, but their specific role in either facilitating substrate diffusion and/or a conformational change at the catalytic site remains to be clarified. Herein, we present crystal structures of cobra (Naja atra) PLA2 in complex with anionic sulfate amphiphiles bound at the interfacial and/or catalytic site; we also determine its orientation against phospholipid membranes using the FTIR method. The results suggest that PLA2 bindings to phospholipid membrane induce a tilting of the hydrocarbon chain of phospholipids with an uneven depth of penetration of aromatic residues of the enzyme into the surface. The interfacial amphiphiles are also shown to diffuse from the interfacial binding site to the active site via binding-induced conformational changes as evidenced by a time-dependent change in the crystal form. Thus, interfacial activation of cobra PLA2 may involve a structural intermediate of the enzyme with interfacially bound amphiphiles to facilitate the diffusion of substrates. The intermediate is suggested to behave similarly to the pre-micellar aggregate of the enzyme and involve the binding of amphiphile to anionic binding cluster region of cobra PLA2 with the hydrocarbon tail of the lipid interacting directly with the hydrophobic amino acid residues located near the N-terminal and the pore region.

並列關鍵字

chloroplast ； GTPase ； TOC ； dimerization ； crystal structure ； GAP ； Arginine finger ； phospholipase A2 ； FTIR ； interfacial binding ； amphiphile binding

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