(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

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研究生：	董容羽 Tung, Jung-Yu
論文名稱：	(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
指導教授：	孫玉珠 Sun, Yuh-Ju
口試委員:
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：	2010
畢業學年度：	99
語文別：	英文
論文頁數：	94
中文關鍵詞：	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
外文關鍵詞：	AnGA, CBM, βCD, G7, GA, PEG, RMSD, Ro, SBD, OMP, ECM, PBS, MAD, ITC, MOPS, ELISA, polyD
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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.

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.

Contents
Chinese abstract    i
English abstract    iii
Contents    v
List of Tables    vii
List of Figures    viii
List of Abbreviations     x

Part I Crystal Structures of Starch Binding Domain of Rhizopus oryzae Glucoamylase Reveal a Polysaccharide Binding Path






Chapter 1 Introduction    1
Chapter 2 Materials and Methods
1.2.1 Protein Expression and Purification    4
1.2.2 Crystallization     4
1.2.3 X-ray Data Collection    5
1.2.4 Structure Determination and Refinement    5
Chapter 3 Results and Discussion
1.3.1 Carbohydrate Binding Module Folding Topology    6
1.3.2 Crystallization    6
1.3.3 X-ray Data Collection and Structure Determination    7
1.3.4 Overall Structures    7
1.3.5 Binding Sites    8
1.3.6 PolyN Loop    10
1.3.7 Binding Affinity for Starch and βCD    11
1.3.8 RoGACBM21 Complexes and Other CBM Superfamilies    13
1.3.9 Liganded and Unliganded RoGACBM21     15
1.3.10 RoGACBM21 Complexes and Other CBM Superfamilies    16

Chapter 4 Conclusion    19
Chapter 5 Tables and Figures    20

Part II Calcium Binds to LipL32, a Lipoprotein from Pathogenic Leptospira, and Modulates Fibronectin Binding
Chapter 1 Introduction     37
Chapter 2 Materials and Methods
2.2.1 Protein Expression and Purification    41
2.2.2 Mass Spectrometry and ICP-AES    42
2.2.3 Size Exclusion Chromatography    42
2.2.4 Analytical Ultracentrifugation    43
2.2.5 Crystallization and X-ray Data Collection     43
2.2.6 Phase Determination, Structural Determination and Refinement    43
2.2.7 Isothermal Titration Calorimetry (ITC)    44
2.2.8 The Strains-all Binding Assay    44
2.2.9 Enzyme-linked immunosorbent ssay (ELISA)    45
2.2.10 Molecular Docking     46

Chapter 3 Results and Discussion
2.3.1 LipL32 Characterization    48
2.3.2 Crystallization     49
2.3.3 MAD Data Collection, Phase Determination and Structure Refinement    49
2.3.4 LipL32 Structure    51
2.3.5 LipL32 is a Ca2+-Binding Protein    52
2.3.6 Conformational changes triggered by Ca2+ binding    53
2.3.7 Ca2+ modulates fibronectin binding to LipL32    56

Chapter 4 Conclusion    59
Chapter 5 Tables and Figures    60
References     86

List of Tables

Part I Crystal Structures of Starch Binding Domain of Rhizopus oryzae Glucoamylase Reveal a Polysaccharide Binding Path

1.1    X-ray diffraction data and refinement statistics of apo SBD and the complex
of SBD-βCD and SBD-G7    20
1.2    Binding affinity of wild-type and mutant RoGACBM21 for starch and βCD    21

Part II Calcium Binds to LipL32, a Lipoprotein from Pathogenic Leptospira, and Modulates Fibronectin Binding

2.1  List of 21 selenium sites found in 5 mol/asu by SOLVE    60
2.2  X-ray diffraction data and Structure Refinement Statistics    61
2.3  Similar three-dimensional structure search of LipL32 by DALI    62
2.4  ICP-AES data of the LipL32    63


List of Figures

Part I Crystal Structures of Starch Binding Domain of Rhizopus oryzae Glucoamylase Reveal a Polysaccharide Binding Path

   Figure 1.1  Structure-based multiple sequence alignment of SBDs    22
   Figure 1.2  Crystals and X-ray diffraction pattern of the RoGACBM21-βCD complex    23
Figure 1.3  Crystals and X-ray diffraction pattern of the RoGACBM21-G7 complex    24
   Figure 1.4  Overall structures of the RoGACBM21 complexes    25
   Figure 1.5  2|Fo|−|Fc| omit electron density map of βCD in the SBD–βCD complex    26
   Figure 1.6  Superimposition of four maltoheptaoses of four molecules per
asymmetric unit in the RoGACBM21–G7 complex    27
   Figure 1.7  Two polyN loops in the SBD–βCD complex    28
   Figure 1.8  Two polyN loops of loop β34 and loop β78 in the SBD–βCD complex    29
   Figure 1.9  Continuous polysaccharide-binding path in the SBD–βCD complex    30
   Figure 1.10  The solvent-accessible surface of the SBD–βCD complex    31
Figure 1.11  Structural superimposition of unliganded and liganded RoGACBM21s    32
Figure 1.12  Structural comparison of RoGACBM21 with other CBM superfamilies are presented in stereo view    33
Figure 1.13  Structural superimposition of RoGACBM21 (PDB code: 2V8L, green) and AnGACBM20 (PDB code: 1AC0, pink) complexes with βCD are presented in stereo view
    34
Figure 1.14  Structural superimposition of RoGACBM21 (PDB code: 2V8L, green) and TvAICBM34 (PDB code: 1UH4, blue) complexes are presented in stereo view    35
Figure 1.15  The molecular packing in the SBD-βCD complex    36





Part II Calcium Binds to LipL32, a Lipoprotein from Pathogenic Leptospira, and Modulates Fibronectin Binding
Figure 2.1  Cell wall of Leptospires and differential induction of early inflammatory    64
Figure 2.2  Bacterial Lipoprotein Biosynthesis    65
Figure 2.3  Sequence and secondary structure of LipL32    66
Figure 2.4  Mass Spectrometry    67
Figure 2.5  Size exclusion chromatography and sedimentation velocity analytical ultracentrifugation of LipL32    68
Figure 2.6  Sedimentation velocity analytical ultracentrifugation of LipL32    69
Figure 2.7  Selenomethionine-derivative LipL32 and Crystal morphology    70
Figure 2.8  Heavy atoms search by SHELX     71
Figure 2.9  Phasing and density modification by SHELX    72
Figure 2.10  Density modification and auto-model building by RESOLVE    73
Figure 2.11  Ramachandran plot of SeLipL32 crystal structure    74
Figure 2.12  Ca2+-bound LipL32    75
Figure 2.13  Ca2+-binding site of LipL32    76
Figure 2.14  Calcium binding affinity for LipL32 determined by ITC    77
Figure 2.15  Ca2+ bind to LipL32 was detected by the Circular Dichroism spectra of stains-all signal    78
Figure 2.16  Structural comparison between Ca2+-bound and Ca2+-free LipL32    79
Figure 2.17  Major conformational change around loop β8β9 between Ca2+-bound and Ca2+-free LipL32    80
Figure 2.18  Possible collagen-binding sites on LipL32    81
Figure 2.19  Interaction between the fibronectin fragment F30 and LipL32    82
Figure 2.20  ELISA of the interaction between F30 and Ca2+-bound or Ca2+-free LipL32    83
Figure 2.21  Model of the LipL32-F30 complex    84
Figure 2.22  Possible hydrogen bonds of the LipL32-F30 complex    85
Figure 2.23  Electrostatic surface potentials of the LipL32-F30 complex    86

                                

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