(一)米根黴糖澱粉酶澱粉結合區域的晶體結構與多醣結合路徑之研究; (二)致病性鉤端螺絲體之脂蛋白LipL32與鈣離子及纖維連接蛋白的結合能力之研究

Crystal Structures of Starch Binding Domain of Rhizopus oryzae Glucoamylase Reveal a Polysaccharide Binding Path; Calcium Binds to LipL32, a Lipoprotein from Pathogenic Leptospira, and Modulates Fibronectin Binding

董容羽(Jung-Yu Tung)

指導教授 ： 孫玉珠

國立清華大學/生命科學院/生物資訊與結構生物研究所/博士(2010年)

摘要

葡萄糖澱粉酶(glucoamylase)水解澱粉和多醣變成葡萄糖。米根黴糖澱粉酶（Rhizopus oryzae GA）由兩個功能區段組成，胺端澱粉結合結構區段(starch-binding domain SBD)和羧端催化結構區段(catalytic domain)，這兩個區段以O型配醣體連結區域連接。RoGA屬於醣類結合模組(carbohydrate-binding modules, CBM)中的第21族（RoGACBM21）。在本研究中,決定米根黴糖澱粉酶澱粉結合區段以及與複合物β-環形糊精(β-CD)及麥芽七糖(maltoheptaose) 的晶體結構。有兩個受體結合位置，結合位置I（色胺酸47）和結合位置II（酪胺酸32），而從我們的研究發現這兩個糖類結合位置是相互作用的。除了疏水性的作用力外，兩段獨特的多天門冬胺酸區域(polyN)包含了連續的天門冬胺酸參與糖結合。酪胺酸32在有受體結合的情況下產生結構變化。為了闡明與多醣結合的作用機制，根據複合物的晶體結構，製造突變的蛋白且分析其與糖類的結合親和力。除了兩個受體結合位置外，長鏈多醣可能會通過酪胺酸67和酪胺酸93，因此提出可能的多醣結合路徑。
Glucoamylase hydrolyzes starch and polysaccharides to β-D-glucose. Rhizopus oryzae glucoamylase (RoGA) consists of two functional domains, an N-terminal starch binding domain (SBD) and a C-terminal catalytic domain, which are connected by an O-glycosylated linker. The SBD of RoGA belongs to the carbohydrate binding modules (CBMs) family 21 (RoGACBM21). The crystal structures of SBD and the complexes with β-cyclodextrin, a cyclic carbohydrate, and maltoheptaose, a linear carbohydrate, were determined. Two carbohydrate binding sites, I (Trp47) and II (Tyr32), were resolved and their binding are cooperative. Besides the hydrophobic interaction, two unique polyN loops comprising consecutive asparagines also participate in the sugar binding. A major conformational change in Tyr32 was observed between unliganded and liganded SBDs. To elucidate the mechanism of polysaccharide binding, a number of mutants were constructed and characterized by quantitative binding isotherm and Scatchard analysis. In addition to sites I and II, a continuous ligand binding surface through Tyr67 and Tyr93 might be essential for long-chain polysaccharides, hence a binding path for RoGA was proposed.

關鍵字

米麴菌澱粉酶 ； 碳水化合物結合模組 ； 環形糊精 ； 麥芽七糖 ； 澱粉酶 ； 聚乙二醇 ； 根均方偏差 ； 米根黴 ； 澱粉結合區塊 ； 外膜蛋白 ； 細胞外間質 ； 磷酸鹽緩衝液 ； 多波長異常散射法 ； 恆溫滴定微量熱儀 ； 丙磺酸 ； 酵素免疫分析法 ； 多重天門冬氨酸

並列摘要

腎小管間質炎(Tubulointerstitial nephritis)是鉤端螺旋體病(leptospirasis)的主要腎臟表現。LipL32位於致病性鉤端螺旋體的外膜上，是一個重要的致病因子以及主要的脂蛋白。它藉由辨認和附著宿主細胞的細胞外間質成分來迴避免疫反應。我們解出與鈣離子結合的LipL32的晶體結構而且其解析度達 2.3埃。LipL32有一個獨特的polyD序列，由七個天冬胺酸殘基序列所構成，在LipL32結構的表面上形成了一個連續酸性的區域而能與鈣離子結合。鈣離子與LipL32結合產生重要的構形變化。利用等溫滴定量熱儀(Isothermal Titration Calorimeter)偵測出鈣離子與LipL32的結合親和力。利用圓二色光譜儀(Circular Dichroism)以及酵素免疫分析法(ELISA)測出纖維連接蛋白與LipL32的結合力。LipL32與纖維連接蛋白之間的相互作用可能與鈣離子結合有關。根據鈣離子結合的LipL32晶體結構以及功能實驗分析，纖維連接蛋白可能的結合區靠近polyD序列。因此鈣離子可能是鉤端螺旋體和宿主細胞的細胞外間質之相互作用的一個重要因子。
Tubulointerstitial nephritis is a cardinal renal manifestation of leptospirosis. LipL32, a major lipoprotein and a virulence factor, locates on the outer membrane of the pathogen Leptospira. It evades immune response by recognizing and adhering to extracellular matrix components of the host cell. The crystal structure of the Ca2+-bound LipL32 was determined at 2.3 A resolution. LipL32 has a novel polyD sequence of seven aspartate residues that forms a continuous acidic surface patch for Ca2+ binding. A significant conformational change was observed for the Ca2+ bound form of LipL32. The calcium binding to LipL32 was determined by ITC. The binding of fibronectin to LipL32 was observed by Stains-all circular dichroism and ELISA experiments. The interaction between LipL32 and fibronectin might be associated with Ca2+ binding. Based on the crystal structure of the Ca2+-bound LipL32 and the Stains-all results, fibronectin probably binds near to the polyD region on LipL32. The Ca2+ binding to LipL32 might be important for Leptospira to interact with the extracellular matrix interaction of the host cell.

並列關鍵字

AnGA ； CBM ； βCD ； G7 ； GA ； PEG ； RMSD ； Ro ； SBD ； OMP ； ECM ； PBS ； MAD ； ITC ； MOPS ； ELISA ； polyD

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