CCL5 (又稱RANTES, Regulated upon Activation expressed by Normal T cells and presumably Secreted）是一種化學趨化素，能調節活化和驅使白血球到達發炎位置引起免疫反應。CCL5受到生理環境和結合目標物影響，以不同聚集形態執行生理功能。單體CCL5能結合接受體活化細胞內訊息傳遞路徑，而CCL5多聚體能結合細胞表面多糖類 (glycosaminoglycan)引起細胞遷移。之前研究解析了CCL5三聚體結構，由兩個突變蛋白質CCL5 E66S和一個野生型CCL5構成。在此結構中，12FAY14區域參與構成交互界面 (interface)。通過序列比對發現， 12FAY14在多種哺乳類動物CCL5家族中具有高度一致性，我們推測這個保留性區域參與CCL5聚集過程。在本研究中，結合核磁共振實驗，我們發現F12和Y14影響CCL5雙聚體的穩定性，Y14還參與了CCL5沉澱，尤其突變蛋白質CCL5-12AAA14能在溶液中穩定維持低聚集形態。我們用結晶學方式解析了CCL5-12AAA14的雙聚體結構，CCL5-12AAA14和野生型CCL5雙聚體相比在N端的交互界面發生骨架位移。我們亦用核磁共振實驗證實了在溶液中這一新雙聚體的存在，確定了F12和Y14影響和改變CCL5的結構特性。我們利用T1, T2, NOE實驗探討CCL5-12AAA14在溶液中的動力學特性，發現其在溶液中同時存在單體和雙聚體，且以雙聚體為主。對CCL5聚集機制的研究將幫助我們了解其結構和生理功能之間的關係，對於研發類似物作為拮抗劑有進一步的幫助。

CCL5 is known to regulate immune responses by mediating the activation and chemotaxis of leukocytes. In order to perform variable functions, CCL5 forms different oligomers depending on environment and binding target. Monomeric CCL5 possesses the ability to activate intracellular signaling upon binding G protein-coupled receptor, while oligomeric CCL5 is capable to trigger protein tyrosine kinase pathway and cause cell migration through cell surface glycosaminoglycans. We determined a CCL5 trimer structure that one additional CCL5 is asymmetrically in complex with CCL5 CC-type dimer. The structure revealed that 12FAY14 sequence was responsible for conjugating the CCL5 dimer. We noticed that the 12FAY14 sequence is conserved in mammalian CCL5s and might be involved in CCL5 oligomerization. By NMR analysis, both residues Phe 12 and Tyr 14 were found to affect the stability of CCL5 dimer whereas Tyr 14 particularly participate in the CCL5 precipitation. The CCL5-12AAA14 variant with double mutations of F12A and Y14A showed a highly impaired oligomerization property. Different NMR methods including 15N T1, T2, and NOE measurements were used to characterize the oligomeric status of CCL5-12AAA14 in solution. The structure of CCL5-12AAA14 was determined by X-ray crystallography and confirmed by NMR. We conclude that CCL5-12AAA14 is deficient in forming oligomer and the major formations in solution are monomer and dimer. Thus, the 12FAY14 sequence in CCL5 not only involves in the oligomerization but also modulates the monomer-dimer equilibrium. The study provides a new insight to understand CCL5 oligomerization and concept for designing CCL5 analog to regulate CCR5 function.

Abstract
Figure Content iii
Table Content vi
Chapter 1 Introduction 1
1.1 Chemokine and its biological function 1
1.2 The interaction of chemokine and receptor 2
1.3 The interaction of CCL5 and glycosaminoglycan (GAG) 3
1.4 The structure of CCL5 and its oligomerization properties 5
1.5 Aim of this study 7
Chapter 2 Materials and Methods 9
2.1 Cloning and mutagenesis 9
2.2 Protein expression and purification 9
2.3 The turbidity assay 10
2.4 Size exclusion chromatography 10
2.5 Crystallization and diffraction data collection 11
2.6 NMR HSQC spectroscopy 12
2.7 NMR relaxation experiments 12
2.8 NMR backbone assignment 12
Chapter 3 Results 14
3.1 Wild-type CCL5 pH-dependent aggregation 14
3.2 The oligomerization and aggregation properties of CCL5 variants 15
3.3 The tertiary fold of CCL5-12AAA14 is sensitive to temperature and salt 17
3.4 Crystal structure of CCL5-12AAA14 17
3.5 CCL5-12AAA14 backbone assignment 19
3.6 The interaction between CCL5-12AAA14 and sulfate ion 22
3.7 Relaxation data analysis 23
3.8 Oligomeric state identification of CCL5 variants 25
Chapter 4 Discussion 26
4.1 Mutant CCL5-12AAA14 26
4.2 Respective roles of F12 and Y14 in CCL5 26
4.3 The FAY sequence in chemokines 28
4.4 The interaction of sulfate ion and CCL5-12AAA14 29
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