趨化素是一類分泌性小型蛋白質，透過與G蛋白受體互動，調控免疫細胞的趨化，參與免疫反應。CXCL9、10和11被歸納為一組，專一性結合CXCR3受體，引導特定免疫細胞遷移到發炎部位。這三種IFN-γ誘導的CXCR3配體具有相似特徵，包括三條反向平行的beta摺板和一個C端alpha-螺旋。儘管它們在序列上有接近40％的相似性，但仍存在許多功能上的區別。為了瞭解它們互動的特殊性，這項研究著眼於CXCR3在細胞外的區域，包括N末端區域和三個胞外環狀區域（ECLs）。這些胞外區域對於配體的選擇性至關重要，有助於理解各趨化因子的結合過程。我們的目標是確定CXCR3各胞外區域在結合各配體時的重要性。為了獲得相關資訊，我們製備了15N標記的CXCL9、10和11進行核磁共振（NMR）測定，同時化學合成了CXCR3的N末端區域、ECL1、2和3，進行滴定實驗。NMR滴定實驗通過檢測HSQC上的化學位移變化，提供了有關結合位點和親和力的重要訊息。我們相信，深入研究CXCL9、CXCL10、CXCL11和CXCR3的結合機制有助於開發調節免疫反應的新型免疫治療策略。

Chemokines, a group of secreted small proteins, regulate immune cell chemotaxis through interaction with 7-transmembrane G protein-coupled receptors, participating in immune responses. CXCL9, 10, and 11 are categorized as a group that specifically binds to the CXCR3 receptor, guiding certain immune cells to inflammatory sites. These three IFN-γ-induced CXCR3 ligands share common features and a generally analogous protein structure.
Despite a nearly 40% sequence identity, notable functional differences persist among them. To unravel the specificity of their interactions, this study focuses on the binding to the extracellular regions of CXCR3, encompassing the N-terminal region and three extracellular loops (ECLs). These extracellular domains play a role in ligand binding specificity, contributing to the understanding of the binding process of different chemokines. Our objective is to elucidate the significance of each CXCR3 extracellular region in binding individual ligands. To acquire pertinent information, 15N-labeled CXCL9, 10, and 11 are prepared for nuclear magnetic resonance (NMR) measurements. Simultaneously, the N-terminal region, ECL1, 2, and 3 of CXCR3 are chemically synthesized for titration experiments. NMR titration experiments, detecting chemical shift perturbations on the HSQC, provide insights into binding sites and affinity. An in-depth investigation into the binding mechanisms of CXCL9/CXCL10/CXCL11 and CXCR3 holds promise for developing novel immunotherapeutic strategies that can modulate immune responses.

Abstract i
中文摘要 ii
Abbreviations iii
Contents iv
Chapter 1: Introduction 1
1.1 Chemokines (Chemotactic cytokine) 1
1.1.2 Subfamilies of Chemokines 1
1.1.3 Structural Basis of Chemokine 3
1.1.4 CXCL9 (Mig) 4
1.1.5 CXCL10 (IP-10) 5
1.1.6 CXCL11 (I-TAC) 6
1.2 CXCR3 8
1.2.1 Structure and Variants of CXCR3 8
1.2.2 CXCR3-Ligand Interactions 8
1.3 Research Strategy 10
Figure 1.1 Subfamilies of Chemokines 13
Figure 1.2 General Structure of CXC-Type Chemokines 14
Figure 1.3 Diverse Oligomeric Structures of Chemokines 15
Figure 1.4 Monomeric Structure of CXCR3-Binding Chemokines 16
Figure 1.5 Structures of CXCL10 17
Figure 1.6 GPCR Topology and CXCR3 Variants 18
Figure 1.7 Two-site/Two-step Model of Chemokines and Their Receptors 19
Figure 1.8 CXCR3 Topology and Associations with CXCL9, 10, and 11 20
Figure 1.9 Multiple Sequence Alignment for Recombinant Chemokines 21
Chapter 2: Materials and Methods 22
2.1 Fundamental Information of Three Recombinant Proteins 22
2.1.1 Expression and Purification of CXCL91-73 23
2.1.2 Expression and Purification of CXCL10 and CXCL11 25
2.1.3 Recombinant protein isotope labeling 27
2.2 Small-Angle X-ray Scattering (SAXS) 27
2.3 NMR Spectroscopy Backbone Assignment 28
2.4 Peptide Design and Synthesis 28
2.5 NMR Titration 29
Table 2.1 Fundamental Information of the Recombinant Proteins 31
Table 2.2 CXCR3-Derived Peptides for Titration Experiments 33
Chapter 3: Experimental Results 35
3.1 Expression and purification of recombinant proteins 35
3.1.1 Expression and purification of recombinant CXCL91-73 35
3.1.2 Expression and purification of recombinant CXCL10 35
3.1.3 Expression and purification of recombinant CXCL11 37
3.2 Structural Analysis of CXCL91-73 38
3.2.1 NMR Backbone Assignment of CXCL91-73 38
3.2.2. Secondary structure of CXCL91-73 39
3.3 Structural Analysis of CXCL10 and CXCL11 39
3.3.1 HSQC Peak Abundance Anomaly 39
3.3.2 Dimeric Peak Investigation HSQC 40
3.3.3 NMR-SAXS Structural Validation 41
3.3.4 NMR Spectroscopy Backbone Assignment 42
3.3.5 Secondary Structure and Disulfide Bond Analysis 43
3.4 CXCL9-CXCR3 Binding Region Analysis 44
3.5 CXCL10-CXCR3 Binding Region Analysis 44
3.5.1 CXCL10-CXCR3 Interaction: HSQC Analysis 45
3.5.2 CXCL10-CXCR3 Interaction: Chemical Shift Perturbation 45
3.5.3 CXCL10-CXCR3 Interaction: Structural Mapping 46
3.6 CXCL11-CXCR3 Binding Region Analysis 47
3.6.1 CXCL11-CXCR3 Interaction: HSQC Analysis 47
3.6.2 CXCL11-CXCR3 Interaction: Chemical Shift Perturbation 48
3.6.3 CXCL11-CXCR3 Interaction: Structural Mapping 49
Figure 3.1 Expression and Purification Profiles of Recombinant CXCL91-73 51
Figure 3.2 Expression and Purification Profiles of Recombinant CXCL10 52
Figure 3.3 Expression and Purification Profiles of Recombinant CXCL11 53
Figure 3.4 Backbone Assignment of CXCL91-73 54
Figure 3.5 Secondary Structure Trends for CXCL91-73 and CXCL9 55
Figure 3.6 HSQC Analysis of CXCL10 at Varying Concentrations 56
Figure 3.7 Concentration-Dependent Analysis of CXCL10 Oligomerization 57
Figure 3.8 SAXS Fitting Curves of CXCL10 58
Figure 3.9 Backbone Assignment of CXCL10 59
Figure 3.10 Trends in the Secondary Structure of CXCL10 60
Table 3.1 13Cβ Chemical Shifts of CXCL10 60
Figure 3.11 HSQC Analysis of CXCL11 at Varying Concentrations 61
Figure 3.12 Concentration-Dependent Analysis of CXCL11 Oligomerization 62
Figure 3.13 SAXS Fitting Curves of CXCL11 63
Figure 3.14 Backbone Assignment of CXCL11 64
Figure 3.15 Trends in the Secondary Structure of Aligned CXCL11 Sequences 65
Table 3.2 13Cβ Chemical Shifts of CXCL11 65
Figure 3.16 Holistic HSQC Spectra of CXCL91-73 Titration with Four CXCR3-derived Peptides 67
Figure 3.17 In-Depth Examination HSQC Spectra of CXCL91-73 Titration with Four CXCR3-Derived Peptides 69
Figure 3.18 Holistic HSQC Spectra of CXCL10 Titration with Four CXCR3-derived Peptides 71
Figure 3.19 In-Depth Examination HSQC Spectra of CXCL10 Titration with Four CXCR3-Derived Peptides 73
Figure 3.20 CSPs of the 1HN and 15N Resonances of CXCL10 upon Binding the Four CXCR3 Peptides 74
Figure 3.21 Mapping CXCR3 N’-Induced CSP at a 1:1 Molar Ratio onto the Crystal Structure of CXCL10 75
Figure 3.22 Spatial Mapping of CXCR3 N'-Induced CSP on CXCL10 76
Figure 3.23 Comparative Analysis of Surface Electrostatic Potential Reveals Likely Binding Region for CXCL10 and CXCR3 N' Peptide 77
Figure 3.24 Holistic HSQC Spectra of CXCL11 Titration with Four CXCR3-Derived Peptides 79
Figure 3.25 In-Depth Examination HSQC Spectra of CXCL11 Titration with Four CXCR3-Derived Peptides 81
Figure 3.26 CSP of the 1HN and 15N resonances of CXCL11 upon Binding the Four CXCR3 Peptides 83
Figure 3.27 Mapping CXCR3 N’-Induced CSP at a 1:1 Molar Ratio onto the NMR Structure of CXCL11 84
Figure 3.28 Comparative Analysis of Surface Electrostatic Potential Reveals Likely Binding Region for CXCL11 and CXCR3 N' Peptide 85
Chapter4: Discussion 86
4.1 Structure of CXCL91-73 and CXCL9 86
4.2 The Interaction between CXCL9 and CXCR3 86
4.3 Structure of CXCL10 87
4.4 The Interaction between CXCL10 and CXCR3 88
4.5 Structure of CXCL11 90
4.6 The Interaction between CXCL11 and CXCR3 92
4.7 Ligands-CXCR3 binding model 93
Figure 4.1 The Binding Site of CXCL9, 10, and 11 for CXCR3 N’ 95
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