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系統識別號 U0026-2103201216383600
論文名稱(中文) 研究馬來腹蛇蛇毒蛋白突變株與其衍生物的結構,動力學與功能的關聯性:揭示其與整合蛋白的交互作用
論文名稱(英文) Structure, Dynamics, and Function Relationships of Rhodostomin Mutants and Variants: Insight into their Interactions with Integrins
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 100
學期 2
出版年 101
研究生(中文) 許家豪
研究生(英文) Jia-Hau Shiu
學號 S5895117
學位類別 博士
語文別 英文
論文頁數 132頁
口試委員 口試委員-余靖
口試委員-符文美
口試委員-陳金榜
口試委員-任卓穎
口試委員-謝奇璋
口試委員-鄭宏祺
口試委員-王淑鶯
指導教授-莊偉哲
中文關鍵字 整合蛋白  去整合蛋白  馬來蝮蛇蛇毒蛋白  核磁共振學  蛋白質結構  蛋白質動力學 
英文關鍵字 integrin  disintegrin  rhodostomin  NMR  structure  dynamics 
學科別分類
中文摘要 整合蛋白(integrin)是一群由α及β次單元體所形成的穿膜異構雙體蛋白,它們表現在細胞的表面使細胞具有黏著能力且媒介細胞與細胞或細胞與細胞間質的交互作用。它們參與許多細胞基本的表現行為,也牽涉到許多疾病的發生;包括腫瘤新生、癌細胞轉移、免疫功能缺失、局部缺血後充血性傷害、病毒感染、骨質疏鬆症及凝血功能不全症。因此整合蛋白被視為有潛力發展藥物設計的目標物。我們的研究中利用一個含有48PRGDMP5序列的去整合蛋白(disintegrin)-馬來蝮蛇蛇毒蛋白(Rhodostomin, Rho),當作骨架來研究去整合蛋白與整合蛋白間的作用關係。利用核磁共振圖譜與蛋白交互作用嵌合程式來研究馬來蝮蛇蛇毒蛋白突變株的結構、動力學與功能間的相關性,並了解特定突變株與不同整合蛋白作用間的結構變化需求,進而了解兩者間的辨識機制。D51E突變蛋白在抑制整合蛋白能力與Rho相比有1000倍以上的差異,為一個較差活性的蛋白,而我們的結構研究發現在Rho中的D51胺基酸其骨架氨基和碳基原子會與整合蛋白的胺基酸R214和R216結合形成重要的氫鍵作用,然而在D51E突變蛋白與integrin的複合物結構中則沒有這樣的氫鍵作用力,所以我們的研究顯示骨架原子以及側鏈原子皆是重要的作用力來與整合蛋白做結合。P48A突變蛋白則是增加4.4倍對整合蛋白α5β1的抑制能力,結構上P48A與Rho具有相同的RGD迴圈結構,結構嵌合分析中也顯示兩者間與整合蛋白α5β1作用位點上並沒有差異,然而在動力學分析上則有所不同,P48A 突變株的S2數值與Rho相比在R49、G50和D51殘基上分別有29%、14% 和28%的降低。在Rex數值上Rho的R49與D51殘基分別為0.91 s1 和1.42 s1,但是在P48A突變株的這些殘基則不具Rex,另外在τe數值上,Rho的R49與D51殘基與P48A相比分別有9.5和5.1倍的降低。這樣結果指出在RGD區間N端Proline殘基的改變會影響其功能,且RGD區間動力學的性質變化也是一個重要因子來影響對整合蛋白α5β1的作用。G50L突變蛋白是一個針對整合蛋白αvβ3具有專一性結合的突變蛋白,根據G50L突變蛋白與整合蛋白的複合物嵌合結構,我們發現L50胺基酸可以容納在整合蛋白αvβ3上所發現的一個袋口區域,相反的在整合蛋白αIIbβ3則沒有發現這樣的一個袋口,因為在αIIbβ3上有一個氫鍵的結合作用介於αIIb與β3次單元之間而妨礙了L50胺基酸的結合。而且我們也發現了G50L的突變亦會改變其RLD區間的彈性力而變得較具剛性,並且也在RLD區間周圍的胺基酸顯現較多構型變化參數,這樣的發現說明了在RLD區間的慢速構型運動會去調節其專一辨識整合蛋白的結合能力。此外經由我們在結構、動力學與功能間的研究,我們開發出一個具高效能專一辨識整合蛋白αvβ3的突變株ARLDDL,在ARLDDL的結晶結構中我們得知其三級結構是與過去已發表的去整合蛋白的結構相近,較大不同處在於其RLD motif有形成較紮實的β轉折結構使得R49與D52的Cα原子間的距離僅有5.5 Å。為了要將ARLDDL當作治療的藥物,其衍生物HSA(C34S)-ARLDDL與PEGylated ARLDDL被開發出來增長其半生期以及降低其免疫原性,然而為了找到這些衍生物的最佳藥物劑型化組成,因此他們在溶液中的物理性質行為是必須被了解與確認。在分子篩光散射實驗中,我們確認了ARLDDL是以一個單體的狀態存在於溶液中,HSA(C34S)-ARLDDL則含有3%的雙體存在,而在PEGylated ARLDDL中發現有8%的多重態PEG的複合物。在熱微差掃描分析儀實驗中,ARLDDL相較於PEGylated ARLDDL具有較高的熱穩定度其熔融熱(Tm)分別為87與56 ℃,相對地HSA(C34S)-ARLDDL則具有兩個熔融熱溫度67與75 ℃。以不同的緩衝液來調整蛋白的穩定度實驗中,我們發現20 mM N-Acetyl-DL-tryptophan and 20 mM sodium caprylate (Octanoate)可以提高HSA(C34S)-ARLDDL的熔融熱達88℃。最後,這些在馬來蝮蛇蛇毒蛋白突變株的結構,功能與動力學的研究以及ARLDDL與其衍生物在溶液狀態下的特性分析,將會幫助我們進一步的把我們的研究運用到臨床藥物的治療。
英文摘要 Integrins are α/β heterodimeric transmembrane proteins that mediate cell-cell and cell-extracellular matrix interactions. They are linked to many pathological conditions, including tumor progression, thrombosis, immune dysfunction, inflammation, and osteoporosis. Therefore, they are attractive therapeutic targets for many human diseases. In this dissertation, I used rhodostomin (Rho), a disintegrin containing a 48PRGDMP53 motif, as a scaffold to study the interactions between integrins and disintegrins. Using nuclear magnetic resonance (NMR) spectroscopy and molecular docking technique, I determined the structure-function-dynamics relationships of Rho and its mutants and identified their structural requirements for integrin recognition. Rho D51E mutant was > 1000 times less active than Rho when inhibiting integrins. The docking of Rho into integrin αvβ3 showed that the backbone amide and carbonyl groups of the D51 residue of Rho formed hydrogen bonds with the integrin residues R216 and R214, respectively. In contrast, these hydrogen bonds were absent in the D51E mutant-integrin complex. Our findings suggest that the hydrogen bond interactions between both the sidechain and backbone of the D51 residue of Rho and integrin are important for their binding. We found that Rho P48A mutant 4.4 times more actively inhibited integrin α5β1. Structural analysis showed that it has a similar 3D conformation for the RGD loop with Rho. Docking analysis of Rho and P48A mutant also showed no difference between their interactions with integrin α5β1. However, the backbone dynamics of RGD residues were different. The S2 values of the P48A mutant residues R49, G50, and D51 were 29%, 14%, and 28% lower than those of Rho. The Rex values of Rho residues R49 and D51 were 0.91 s1 and 1.42 s1; however, no Rex was found for those of the P48A mutant. The τe values of Rho residues R49 and D51 were 9.5 and 5.1 times lower than those of P48A mutant. These results demonstrate that the N-terminal proline residue adjacent to the RGD motif affect its function and dynamics, which suggests that the dynamic properties of the RGD motif may be important in Rho’s interaction with integrin α5β1. Rho G50L mutant was found to be an integrin αvβ3-specific disintegrin. The docking models of G50L mutant into integrins showed that L50 residue of G50L mutant can be accommodated by a cavity within the interface between αv and β3 subunits. In contrast, no observable cavity was found from integrin αIIbβ3 due to a hydrogen bonds formation between the Y190 residue of αIIb and the R216 residue of β3 subunits, resulting in the blockage of the L50 residue binding. We also found that the G50L mutation increased the rigidity of RLD motif, and the adjacent residues exhibited slow conformational exchange. Our findings reveal that slow motions of the RLD motif also play a vital role in modulating integrin recognition binding. According to their function-structure-dynamics relationships, a potent and selective integrin αvβ3 antagonist, ARLDDL mutant, was designed. 3D structure of ARLDDL mutant was determined by X-ray crystallography, and its tertiary fold is the same with reported disintegrin structures. The only difference was found from the RLD motif of the ARLDDL loop. It had a tight β-turn structure with a distance of 5.5 Å between R49 (Cα) and D52 (Cα) in comparison with those of disintegrins ranging from 6.8-8.4 Å. HSA(C34S)-ARLDDL and PEGylated ARLDDL were engineered to increase half-life and to reduce immunogenicity. To develop them for clinical trials, we characterized their solution behavior and identified their formulation conditions. The analysis of size-exclusion chromatography with multi-angle laser light-scattering (SEC-MALLS) showed that ARLDDL mutant existed as a monomer, HSA(C34S)-ARLDDL contained 3% of dimer form, and PEGylated ARLDDL had 8% of multi-PEG species. In differential scanning calorimetry (DSC) experiments, ARLDDL mutant had higher thermal stability than PEGylated ARLDDL, and their melting temperatures (Tm) were 87℃ and 56℃. In contrast, HSA(C34S)-ARLDDL exhibited two Tm values that were 67℃ and 75℃, suggesting that it formed two conformers under histidine and phosphate buffer systems. We also found that the buffer containing 20 mM N-Acetyl-DL-tryptophan and 20 mM sodium caprylate (Octanoate) could increase the thermal stability of HSA(C34S)-ARLDDL with Tm of 88℃. Our studies on structure-function-dynamics relationships of Rho and its mutants and the characterization of ARLDDL and its variants will bridge our designed integrin drugs from discovery to development and support their future clinical trials.
論文目次 CHINESE ABSTRACT………………………………………………...…… I
ABSTRACT……………………………………………………....................III
ACKNOWLEDGMENT……………………………………………………...V
TABLE OF CONTENTS…………………...……………………….............VI
LIST OF TABLES……………………………………………………….... IX
LIST OF FIGURES……………………………………………………….....X
ABBRIEVATION………………………………………………………..…XII
CHAPTER 1 INTRODUCTION 1
1.1 BACKGROUND 1
1.2 INTEGRINS 2
1.2.1 Overview of integrins 2
1.2.2 Integrins in Cancer 3
1.2.3 Integrin Structures 3
1.3 DISINTEGRINS 4
1.3.1 Overview of disintegrins 4
1.3.2 Biomedical applications of disintegrins 6
1.4 RHODOSTOMIN (RHO) 7
1.4.1 Rhodostomin function 7
1.4.2 Identification of intgerin recognition sequences by mutagenesis study of Rho 7
1.4.3 Potent and selective integrin αvβ3 antagonist-ARLDDL 9
1.4.4 Development of long acting integrin αvβ3-specific proteins 9
1.5 STRUCTURE-BASED TECHNIQUE IN PROTEIN DRUG DEVELOPMENT 11
1.5.1 Nuclear magnetic resonance (NMR) investigations of protein structure and dynamics 11
1.5.2 X-ray crystallography 12
1.5.3 Molecular docking 13
1.6 PREFORMULATION RESEARCH 14
1.6.1 Introduction 14
1.6.2 Size exclusion chromatography with multi-angles laser light scattering (SEC-MALLS) 15
1.6.3 Differential scanning calorimetry (DSC) 16
1.7 RESEARCH MOTIVATION AND SPECIFIC AIMS 17
1.7.1 Structural and dynamic characterization of Rho and its mutants 17
1.7.2 Preformulation study of ARLDDL and its variants 17
CHAPTER 2 METHODS AND MATERIALS 19
2.1 PROTEIN SAMPLES 19
2.1.1 Expression and purification of Rho and its D51E, P48A, and G50L mutants 19
2.1.2 Expression and Purification of ARLDDL 20
2.1.3 Expression and Purification of HSA(C34S)-ARLDDL 20
2.1.4 PEGylation of ARLDDL 21
2.2 NUCLEAR MAGNETIC RESONANCE (NMR) 21
2.2.1 NMR spectroscopy 21
2.2.2 NMR structure calculations 22
2.2.3 Measurements of NMR dynamics 23
2.2.4 ModelFree analysis 24
2.3 MOLECULAR DOCKING 25
2.3.1 Molecular docking of Rho and D51E into integrin αvβ3 25
2.3.2 Molecular docking of Rho and P48A into integrin α5β1 26
2.3.3 Molecular docking of G50L into integrin αvβ3 and αIIbβ3 26
2.4 PROTEIN CRYSTALLOGRAPHY 27
2.4.1 Crystallization of ARLDDL 27
2.4.2 Diffraction data collection, structure determination and refinement 27
2.5 SIZE EXCLUSION CHROMATOGRAPHY WITH MULTI-ANGLES LASER LIGHT SCATTERING (SEC-MALLS) 28
2.6 ANALYTICAL SIZE-EXCLUSION CHROMATOGRAPHY 28
2.7 DIFFERENTIAL SCANNING CALORIMETRY (DSC) 29
CHAPTER 3 RESULTS 30
3.1 SOLUTION STRUCTURE DETERMINATION OF RHO AND ITS D51E, P48A, AND G50L MUTANTS BY NMR 30
3.2 DYNAMIC PROPERTIES OF RHO AND ITS D51E, P48A, AND G50L MUTANTS 31
3.2.1 Dynamics difference between Rho and its D51E mutant 31
3.2.2 Dynamics difference between Rho and its P48A mutant 33
3.2.3 Dynamics difference between Rho and its G50L mutant 34
3.3 DOCKING MODELS OF RHO AND ITS MUTANTS WITH INTEGRINS 35
3.3.1 Interaction difference in the docking models of integrin αvβ3-Rho and -D51E mutant complexes 35
3.3.2 No structural differences between the integrin α5β1 complexes of Rho and the P48A mutant 36
3.3.3 Structural differences between the G50L mutant in complex with integrins αvβ3 and αIIbβ3 37
3.4 CRYSTAL STRUCTURE OF ARLDDL 38
3.4.1 Overall structure of ARLDDL 38
3.4.2 Structural comparison with other disintegrins 39
3.5 DETERMINATION OF MOLECULAR WEIGHT AND OLIGOMERIC STATE IN SOLUTION BY SEC-MALLS 40
3.5.1 System calibration by BSA 40
3.5.2 Characterization of ARLDDL 41
3.5.3 Characterization of PEGylated ARLDDL 41
3.5.4 Characterization of HSA(C34S)-ARLDDL 42
3.6 HYDRODYNAMIC ANALYSIS BY SIZE-EXCLUSION CHROMATOGRAPHY 42
3.6.1 Stokes Radius Determination of ARLDDL, HSA(C34S)-ARLDDL, PEGylated ARLDDL 42
3.7 THERMAL STABILITY MEASURED BY DSC 43
3.7.1 Protein Melting Temperatures in PBS Buffer 43
3.7.2 Melting Temperature of HSA(C34S)-ARLDDL in Different Formulation Buffers 43
CHAPTER 4 DISCUSSION 45
4.1 EFFECT OF D TO E MUTATION OF THE RGD MOTIF IN RHO ON ITS FUNCTION, STRUCTURE, AND DYNAMICS 45
4.2 EFFECT OF P TO A MUTATION OF THE N-TERMINAL RESIDUE ADJACENT TO THE RGD MOTIF IN RHO ON ITS ACTIVITY, STRUCTURE, AND DYNAMICS 46
4.3 EFFECT OF G TO L MUTATION OF THE RGD MOTIF IN RHO ON ITS ACTIVITY, STRUCTURE, AND DYNAMICS 49
4.4 TIGHT TURN STRUCTURE OF RLD MOTIF OF ARLLDL 50
4.5 STRUCTURE-DYNAMICS-FUNCTION RELATIONSHIPS OF RHO MUTANTS 51
4.6 DIFFERENCES OF INTEGRIN RECOGNITION BY ECM AND RHO MUTANTS 52
4.7 SOLUTION BEHAVIOR OF ARLLDL VARIANTS AND POTENTIAL PROBLEM IN FORMULATION RESEARCH 53
CHAPTER 5 CONCLUSIONS 55
REFRENCES…………………...……………………………......................57
TABLES………………………………………………………......................68
FIGURES………………………………………………………...…………86
PUBLICATIONS………………………………………………………......113
APPENDIX………………………………………………………...............114
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