TvCyP1和TvCyP2都是環孢素型的肽基 - 脯氨酸異構酶，它們存在於人體的寄生蟲: 陰道滴蟲中，TvCyP1與轉錄因子Myb1相互作用並協助其進入細胞核，而Myb1、Myb2和Myb3會一起調節ap65-1基因的表達，產生氫化酶體細胞粘附酶，進而導致陰道滴蟲的感染。在這篇研究論文中，TvCyP1及其複合物: Myb1的最小結合序列（Myb1104-111）的X-ray晶體結構被解析出來，其中TvCyP1形成了同型二聚體，如同在水溶液中也觀察到一樣的二聚體現象；在複合物的結構中，一個Myb1104-111分子的P107結合到每個TvCyP1蛋白的活性位點上， NMR數據進一步證明TvCyP1可以催化Myb1104-111中P107的順式/反式異構化，然而，在折疊良好的Myb1蛋白（Myb135-141）中，P107位於由-螺旋結構環繞的簡短環狀結構中，使其難以與TvCyP1的活性位點結合，不過核磁共振研究近一步證明，由於P107周圍的胺基酸皆具有微秒範圍的動態變化，使得Myb135-141和Myb1104-111胜肽類似，也可與TvCyP1的活性位點相互作用。另外，根據結構我找到了兩個在TvCyP1二聚化中至關重要的殘基，並構建了只形成單體的雙突變蛋白（FM-TvCyP1）， 發現Myb135-141與FM-TvCyP1的相互作用顯著減弱，表示TvCyP1的二聚化對於與Myb135-141的相互作用是必需的。這個研究提供了關於TvCyP1-Myb1相互作用的詳細結構分析，可以為設計藥物以治療抗藥菌株提供一個新的方向。

TvCyP1 and TvCyP2 are cyclophilin type peptidyl-prolyl isomerases present in the human parasite Trichomonas vaginalis. TvCyP1 interacts with the transcription factor Myb1 and assists in its nuclear translocation. Myb1 along with Myb2 and Myb3 regulates the expression of ap65-1 gene that encodes for a hydrogenosomal cytoadherence enzyme, thereby leading to pathogenesis. The crystal structures of TvCyP1 and its complex with the minimum TvCyP1-binding sequence of Myb1 (Myb1104-111) reported here shows that, TvCyP1 formed a homodimer as observed in solution. In the complex structure, P107 of one Myb1104-111 molecule was bound to the active site of each TvCyP1 protomer. NMR data further showed that TvCyP1 can catalyze the cis/trans isomerization of P107 in Myb1104-111. However, in the well-folded Myb1 protein (Myb135-141), P107 lies in a short loop surrounded by helices, making it difficult to bind to the active site of TvCyP1. NMR studies showed that similar to Myb1104-111 peptide, Myb135-141 also interacted with the active site of TvCyP1, facilitated by the slow dynamics in Myb135-141 surrounding P107. Identification of the two residues that are crucial in TvCyP1 dimerization led to the construction of a monomeric double mutant (FM-TvCyP1). Myb135-141 showed significantly weakened interaction with FM-TvCyP1, suggesting dimerization of TvCyP1 is essential for interaction with Myb135-141. This study provides detailed structural insights on TvCyP1-Myb1 interaction that could pave the way for newer drugs to treat drug-resistant strains.


 

LIST OF CONTENTS

Acknowledgement....i
Abstract (English)....iii
Abstract (Chinese)....iv
List of Contents....v
List of Abbreviations....vii
Chapter 1 Introduction....1
1.1. Peptidyl prolyl isomerases....1
1.2. Cyclophilins....3
1.2.1. Structural characteristics of cyclophilins....4
1.2.2. Cylophilins in T.vaginalis....6
1.3. T.vaginalis....8
1.3.1. Clinical relevance....8
1.3.2. Myb transcription factors in T.vaginalis....10
1.4. Purpose and outline of this study....11
Chapter 2 Structure and solution behaviour of TvCyP1....15
2.1. Biophysical behaviour of TvCyP1 in solution....15
2.2. X-ray crystal structure of TvCyP1....18
2.3. Dimer interface of TvCyP1....22
2.4. Comparison of the structure of TvCyP1 with other cyclophilins....23
2.5. Effect of mutations of dimer interface residues....25
2.6. FM-TvCyP1: the monomeric double mutant of TvCyP1....25
Chapter 3 Interaction of TvCyP1 with Myb1 peptide....28
3.1. Minimum binding sequence in Myb1....31
3.2. Peptidyl-prolyl isomerase activity of TvCyP1....32
3.3. X-ray crystal structure of TvCyP1 in complex with Myb1 peptide....36
3.4. Interaction of FM- TvCyP1 with Myb1 peptide....40
Chapter 4 Interaction of TvCyP1 with Myb1 protein ....42
4.1. Mapping the TvCyP1 binding site in Myb1 protein by NMR....42
4.2. Identification of Myb1 protein interaction region in TvCyP1 by NMR....48
4.3. Reduction in Myb1 dynamics upon interaction with TvCyP1....56
Chapter 5 Discussion....58
Chapter 6 Conclusion....62
Chapter 7 Materials and methods....64
7.1. Expression and purification of TvCyP1 and TvCyP1 mutants....64
7.2. Preparation of Myb1, Myb1 mutants and Myb1 peptides....65
7.3. SEC-MALS....65
7.4. Analytical ultracentrifugation.... 66
7.5. Fluorescence polarization experiments....66
7.6 Protein crystallization and data collection....66
7.7. Structure determination and refinement....67
7.8. NMR experiments....68
References....71
Appendix ....82
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