臺灣博碩士論文加值系統

本論文第一部份經由Arabidopsis thaliana bi-functional nuclease (AtBFN2)結晶結構探討此蛋白質催化水解單股DNA與RNA的機制，由結構解析了解參與基質催化反應的殘基。阿拉伯芥基因At1g68290可轉譯成雙功能核酸酶AtBFN2 (EC 3.1.30.1)，藉由鋅離子參與催化反應對單股DNA與RNA進行切割反應產生5’ 核苷酸最終產物。AtBFN2 為一醣蛋白，先前文獻研究證明此蛋白質參與植物的分化與發育。在本論文中，我們將C端帶有組胺酸標籤 (his-tag) 的雙功能核苷酸酶之融合蛋白質轉殖於阿拉伯芥中大量表現並純化並成功解析AtBFN2結構，解析度為1.76 A。AtBFN2晶體內一硫酸根離子與催化中心鋅離子藉由氫鍵交互作用。AtBFN2結構由13個α-螺旋、五個短β-平板與4個雙硫鍵組成的蛋白質分子。我們發現催化中心鋅離子與AtBFN2 會與分子內5個組氨酸與3個天門冬氨酸的側鏈、2個來自色氨酸骨架上的原子、以及環境中水分子與硫酸根離子有交互作用。 AtBFN2 分子Asn91、Asn110、Asn184位置被後轉譯修飾作用結合約22個糖分子的醣蛋白。我們進一步將AtBFN2-sulfate-adenine 複合體與已發表P1核酸酶 (P1 nuclease；PDB ID : 1AKO) 的結構比較，發現兩者催化方式皆利用被鋅離子活化的水分子對磷分子做in-line attack方式水解單股DNA與RNA。
本論文第二部分利用結構解析研究台灣龜殼花毒液金屬蛋白酶(snake-venom metalloproteinase) TM-1與內生性三肽間抑制作用。TM-1 屬於P-I 族群金屬蛋白酶，利用結晶學與結構生物學，我們獲得解析度為1.8 A的TM-1 3D結構。TM-1與已知的SVMP特性相似，皆利用三個雙硫鍵穩定其結構，分別位於 Cys119-Cys198、Cys160-Cys182、Cys162-Cys165。其結構活性區為一Zinc ion 被三個histidine 與水分子包圍形成tetrahedral 構形。
我們進一步分析TM-1與另一種同為台灣龜殼花分離出的蛇毒TM-3 的S1’活性區，發現基質特異性與S1’底部的胺基酸殘基變異有關。同時利用水解胰島素B鍊研究S1’活性區對基質切位，發現TM-1 對中型與疏水性殘基有較高的水解能力。

The dissertation is divided into two parts. The first topic is to discuss the structure of Arabidopsis thaliana AtBFN2, a gene participates in development differentiation. The A. thaliana gene At1g68290 was expressed as a C-terminal hexahistidine fusion protein AtBFN2 (EC 3.1.30.1). The bi-functional nuclease AtBFN2 from A. thaliana depends on zinc ion for cleaving single stranded DNA and RNA to yield 5''-nucleotides. It is a glycoprotein that participates in plant development and differentiation. In this study, the crystal structure of AtBFN2 shows a bound sulfate ion in the active site, at the center of the tri-nuclear cluster of zinc ions. The protein folds into a mostly α-helical structure with five short β-strands and contains four disulfide bonds. The zinc ions are coordinated to the side chains of three Asp and five His residues, two backbone atoms of Trp1, the sulfate ion, and a water molecule. An adenine base is bound adjacent to the active site and stacks with Tyr59. The core sugar residues attached to the three N-glycosylation sites of Asn91, Asn110 and Asn184 are also observed. By comparison with the nuclease P1 structure (PDB ID: 1AK0), the AtBFN2-sulfate-adenine complex model suggests a similar catalytic mechanism, in which the reaction starts with in-line attack at the phosphate by a zinc-activated water molecule.
The other topic of this dissertation is to study snake-venom metalloproteinase (SVMP). The crystal structure of TM-1, a P-I class snake-venom metalloproteinase (SVMP) from the Trimeresurus mucrosquamatus venom, was determined at 1.8-A resolution. The overall structure of TM-1 is an oblate ellipsoid that contains three disulfide crosslinks, Cys119-Cys198、Cys160-Cys182、Cys162-Cys165. At active site, one zinc ion is bound to four ligands, including three conserved histidines and one water, displaying a tetrahedral geometry. The active site shows a deep S1’ substrate-binding pocket limited by the non-conserved Pro174 at the bottom. Further comparisons with other SVMPs suggest that the deep S1’ site of TM-1 correlates with its high inhibition sensitivity to the endogenous tripeptide inhibitors. Proteolytic specificity analysis revealed that TM-1 prefers substrates having a moderate-size and hydrophobic residue at the P1’ position, consistent with our structural observation.

Abstract (in Chinese)………………………………………………………………….V
Abstract……………………………………………………………………………...VII
Part A: Structural studies of AtBFN2, a nuclease from Arabidopsis thaliana and its catalytic mechanism
1. Introduction……………………………………..………………….………….......1
2. Materials and methods…………………………………………….………………5
2.1 Transgenic plant growth conditions……………………………………………….5
2.2 Purification of recombinant AtBFN2 from transgenic Arabidopsis thaliana……5
2.3 Crystallization, data collection, and structure determination ……………………..6
3. Results……………………………………………………………………………...8
3.1. Overall structure of AtBFN2……………………………………………………...8
3.2. Active-site configuration………………………………………………………….9
3.3. Carbohydrate structure…………………………………………………………..10
4. Discussion…………………………………………………………………………12
5. Tables and figures………………………………………………………………..18
Fig. 1 Phylogenetic tree of single‐strand-specific nucleases…………………….......18
Fig. 2 Diagram of PcNP1 hydrolyzes DNA, RNA and 3''-monophosphonucleotides..19
Fig. 3 Purification flow chart of AtBFN2……………………………………………20
Fig. 4 Purification of AtBFN2 from transgenic plants……………………………….21
Fig. 5 The AtBFN2 protein crystals formed in different screening conditions….......22
Fig. 6 Diagram of thiophosphorylated nucleotides…………………………………23

Fig. 7 AtBFN2 crystals were improved by adding thiophosphorylated nucleotide….24
Fig. 8 Overall structure of AtBFN2………………………………………………….25
Fig. 9 Structure comparison of AtBFN2 with PcNP1 and BcPLC……………..........26
Fig. 10 The amino-acid sequence aligment of AtBFN2, BcPLC and PcNP1………27
Fig. 11 The active-site structure of AtBFN2……………………………………….28
Fig. 12 The Stereo view of AtBFN2 active site…………………………………….29
Fig. 13 The electron density maps of ligands and sugars of AtBFN2……………….30
Fig. 14 The stereo view of carbohydrate interactions………………………………..31
Fig. 15 Alignment of plant S1/P1-type nucleases.………………………………...32
Fig. 16 Comparison of the active-site bound ligands in AtBFN2 and PcNP1……….33
Fig. 17 The carbohydrate coordinate residues of AtBFN2..........................................34
Fig. 18 Substrate model in the active site of AtBFN2 (a)……………………………35
Fig. 19 Substrate model in the active site of AtBFN2 (b)……………………………36
Fig. 20 Proposed catalytic mechanism of AtBFN2…………………………….......37
Fig. 21 Bonds involved in catalysis………………………………………………….38
Fig. 22 Comparison of the active-site structures of AtBFN2 and SlTBN1………….39
Table 1 Data collection and refinement statistics.………………………………...40

Part B: Structural basis for distinct susceptibilities of snake venom metalloproteinases to the endogenous tripeptide inhibitors

1. Introduction……………………………………..………………………………....41
2. Materials and methods……………………………………………………….......45
2.1 Crystallization……………………………………………………………............45
2.2 Structure determination and refinement………………………………………….46
2.3 Protein N-terminal sequence analysis……………………………………………47
2.4 Proteolytic specificity…………………………………………………………….47
3. Results and discussion……………………………………………………………48
3.1 Crystallographic sequence and overall structure…………………………………48
3.2 The active-site environment……………………………………………………...50
3.3 Implication for sensitivity to the endogenous peptide inhibitors…………...........52
3.4 Proteolytic specificity…………………………………………………………….53
4. Conclusion………………………………………………………………………...54
5. Tables and figures………………………………………………………………55
Fig. 1 Crystal of TM-1 from T .mucrosquamatus used for data collection…………55
Fig. 2 Amino-acid sequence and crystal structure of TM-1………………………….56
Fig. 3 The final refined structure of TM-1 overlaid with the experimental
electron-density map…………………………………………………………………57
Fig. 4 Multiple sequence alignment of TM-1 with other twenty-one SVMPs……….58
Fig. 5 A ribbon diagram in stereo view of the overall structure of TM-1……………60
Fig. 6 Superimposition of TM-1 structure with the structures of other eight P-I SVMPs…………………………………………………………………………….61
Fig. 7 The zinc-binding environment of TM-1 structure………………………….62
Fig. 8 Catalytic mechanism of metalloproteinase…………………………………....63
Fig. 9 Substrate specificity of TM-1………………………………………………....64
Fig. 10 Metal ions bound to the surface of TM-1……………………………………65
Fig. 11 Comparison of the active-site structures of TM-1 and TM-3………………66
Table 1 Comparison of cleavage specificities of various snake-venom matalloproteinases with oxidized insulin B-chain as substrate……………………67
Table 2 Data collection and refinement statistics……………………………………68
Table 3 Inhibition constants for the synthetic analogues of peptide inhibitors……...69
References……………………………………………………………………….......70
Appendix……………………………………………………………….....................76

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