CCL5（又稱RANTES）是人體內一項重要的趨化因子，在發炎反應過程中會趨化和誘發白血球附著及移轉其位置至發炎部位。CCL5與許多重要的疾病有關，不論是慢性發炎或是急性發炎性疾病。先前研究指出，CCL5的多種聚合型式會誘發不同生物反應，如細胞遷移、T細胞活化、細胞凋零和HIV細胞入侵。聚合型式受到多種因子調控，例如濃度、溫度、pH值等，CCL5在生理條件下以多聚體型式展現，但在結構生物學上尚未有大的突破。認識CCL5形成多聚體的機制有助於瞭解誘發化學趨化性的作用方式及其與受體的結合作用。截至目前為止，CCL5在臨床或細胞上之相關研究大多以老鼠做為實驗之動物模型，老鼠與人類CCL5在序列上十分相似，但目前研究並未針對老鼠CCL5做任何結構資訊之發表，因此本研究利用核磁共振技術解出老鼠CCL5雙聚體結構，證明老鼠與人類兩者之間的結構相似性。我們又以雙聚體為基礎單位，配合前人點突變之研究成果，設計出新的策略獲取限定長度的三聚體。CCL5會在中性條件下形成高單位CCL5聚合體並大量沈澱，在酸性條件下則是以雙聚體為主，而E66S在中性則是以雙聚體為主。CCL5與E66S在比例固定且適當的pH下混合，成功地在液相獲取三聚體，並利用小角度X光散射儀和凝膠過濾層析法驗證，再利用蛋白質結晶及X光繞射的技術成功解析出CCL5三聚體的結構，其三維結構顯示A和B鏈形成之雙聚體與以往所知的趨化素CC類雙聚體類似，衍伸出的單體C鏈以新的方式鍵結至另一分子的N端。這特殊的三聚體構型能重複排列並衍生成一條W型長鏈，排列蛋白質表面正電的氨基酸且完整的曝露40s loop（GAG多醣體的結合區域）。我們以穿透式電子顯微鏡進一步證實N端序列移除的蛋白會影響CCL5多聚體形成之能力。本研究解出一組新的多聚體構型之結合區域，為多聚體之研究提供了新的思考方向。

CCL5, also known as RANTES, controls leukocyte migration and is associated with inflammation and development. CCL5 shows great propensity for protein oligomerization in physiological condition. The oligomer formation has been correlated with cell migration, T cell activation, apoptosis, and HIV cell entry. The property highly depends on protein concentration and pH. However, the structural information of the high-order oligomer is still limited. The study of the CCL5 oligomerization mechanism would aid the understanding of how CCL5 oligomer performs chemotaxis and binds cell surface receptors. Using human and mouse CCL5 as models, we determined the CCL5 dimer structure by nuclear magnetic resonance (NMR) and X-ray crystallography, either using native proteins or mutants. The structure study of mouse CCL5 shows good similarity with human CCL5 structure. However, the dimeric interface constituted by the N-terminal ends contains structural flexibility, therefore, causing distinct orientations between the two monomeric units. In addition, although the two proteins have ~84% sequence identity, they have slightly different oligomerization sensitivity toward pH change. The property could be correlated with the functional concentrations. The solved dimeric structure acts as the building block for constructing the oligomer formation. We propose a new strategy to trap the CCL5 trimer. To prepare the trimer formation, we construct a CCL5 mutation, E66S, to eliminate the aggregation propensity that E66S only shows dimerization property. By mixing native CCL5 with the CCL5-E66S mutant under an optimal protein ratio, CCL5 trimer existed as a dominative formation in solution, which is detected by SAXS, FPLC and NMR. The trimer structure is solved by X-ray crystallography. Surprisingly, the N-terminal region contributes additional interaction with the third CCL5 unit. This structure could be extended to a W-sharped oligomer that elicits a new model for CCL5 oligomerization. This oligomer structure possesses many positive charged residues and fully exposes the 40s loop, readily for GAG binding. In the study, we compared with the oligomerization status of the N-terminal truncated CCL5 mutant. The images from transmission electron microscope (TEM) emphasize the significance of CCL5 N-terminus. The new trimer structure defines a novel interface, responsible for CCL5 oligomerization

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