嵌膜型質子傳送焦磷酸水解酶之晶體結構｜國立清華大學博碩士論文庫

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研究生：	林士鳴 Lin, Shih-Ming
論文名稱：	嵌膜型質子傳送焦磷酸水解酶之晶體結構 Crystal Structure of a Membrane-embedded H+-translocating Pyrophosphatase
指導教授：	潘榮隆 Pan, Rong-Long
口試委員:	孫玉珠 Sun, Yuh-Ju 張晃猷 Chang, Hwan-You 張文綺 Chang, Wen-Chi 蕭傳鐙 Hsiao, Chwan-Deng 潘榮隆 Pan, Rong-Long
學位類別：	博士 Doctor
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2013
畢業學年度：	101
語文別：	英文
論文頁數：	43
中文關鍵詞：	膜蛋白、質子傳輸、焦磷酸水解酶、X光蛋白質晶體學、液泡
外文關鍵詞：	Membrane protein, Proton transport, Pyrophosphatase, X-ray protein crystallography, Vacuole
相關次數：	點閱：3 下載：0
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質子傳送焦磷酸水解酶可藉由水解焦磷酸建立跨生物膜的質子梯度，為一種主動傳輸型質子傳送蛋白。此酵素主要以雙次體的形式存在於植物液泡膜與一些原核生物的細胞膜中。此酵素之三維結構仍尚未解構，其酵素反應與質子傳送機制也尚待釐清。此研究描述綠豆質子傳送焦磷酸水解酶之晶體結構，此結構為酵素與亞氨二磷酸鹽(imidodiphosphate)之結合狀態，其解析度為2.35埃。每個綠豆質子傳送焦磷酸水解酶次體由十六根穿膜螺旋形成一個穿膜區間。每個次體利用數個帶電胺基酸與五個鎂離子一將個亞氨二磷酸鹽分子牢牢固定在其細胞質側。我們發現一個嶄新的質子傳送途徑由六根核心穿膜螺旋中心組成。質子傳送可藉由水解焦磷酸而啟動，進而經由一條由Arg242, Asp294, Lys742 和Glu301所組成的途徑傳送到液泡膜內腔。本論文總結提出一個運作模型用以解釋此酵素之焦磷酸水解與質子傳送知耦合關聯機制。

H+-translocating pyrophosphatases (H+-PPases) are active proton transporters that establish a proton gradient across the endomembrane by means of pyrophosphate (PPi) hydrolysis. H+-PPases are found primarily as homodimers in the vacuolar membrane of plants and the plasma membrane of several protozoa and prokaryotes. The three-dimensional structure and detailed mechanisms underlying the enzymatic and proton translocation reactions of H+-PPases are unclear. Here we report the crystal structure of a Vigna radiata H+-PPase (VrH+-PPase) in complex with a non-hydrolysable substrate analogue, imidodiphosphate (IDP), at 2.35 Å resolution. Each VrH+-PPase subunit consists of an integral membrane domain formed by 16 transmembrane helices. IDP is bound in the cytosolic region of each subunit and trapped by numerous charged residues and five Mg2+ ions. A previously undescribed proton translocation pathway is formed by six core transmembrane helices. Proton pumping can be initialized by PPi hydrolysis, and H+ is then transported into the vacuolar lumen through a pathway consisting of Arg 242, Asp 294, Lys 742 and Glu 301. We propose a working model of the mechanism for the coupling between proton pumping and PPi hydrolysis by H+-PPases.

Contents
Introduction    1
Materials and Methods    5
Cloning, expression and purification    5
PPi hydrolysis activity assay    6
Crystallization and X-ray data collection     6
Structural determination and refinement    7
Results and Discussion    8
The overall structure of VrH+-PPase    8
The substrate/IDP binding pocket    9
The proton translocation pathway    11
A proposed enzymatic mechanism model    12
Future prospects    14
References    16
Tables    23
Table 1, Data collection, phasing and refinement statistics    24
Table 2, Detailed interactions of dimer interface    26
Table 3, The binding interaction of IDP and Mg2+ ions     29
Table 4, The salt bridge network near the active site    30
Table 5, PPi hydrolysis and proton pumping activities of variants with mutation at residues along proton translocation pathway     31
Figures    32
Figure 1, Multiple sequence alignment of H+-PPase    33
Figure 2, The crystal structure of the VrH+-PPase-IDP complex    36
Figure 3, Structural topology of the VrH+-PPase    37
Figure 4, Dimer interface of the VrH+-PPases    38
Figure 5, The substrate binding site    40
Figure 6, The salt bridge network near the active site    41
Figure 7, The proton transport pathway of VrH+-PPase    42
Figure 8, A working model for proton pumping in VrH+-PPase    43

                                

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