CXCL4是由人血小板分泌的CXC型趨化因子，它參與多種生理功能，包括凝血、細胞趨化性和抗血管生成。CXCL4L1是CXCL4的一個非等位變異體，與CXCL4的區別僅在於C端α螺旋中的三個胺基酸。此三個胺基酸的差異導致兩者C端α螺旋的方向不同，CXCL4L1的C端螺旋方向是向外伸展，使CXCL4L1傾向從四聚體解離為單體。根據先前研究顯示，CXCL4和CXCL4L1是透過單體的形式激活CXCR3並誘發生理功能。因此，我們建構了兩個單體突變體，CXCL4-E28K-V29P-K31P和CXCL4L1-E28K，讓趨化因子在中性條件下保持單體狀態。我們測試了突變體與的CXCR3不同膜外區域片段間的作用，包括N端延伸區和第二胞外環。NMR滴定實驗僅證明了N端硫酸化延伸區具有趨化因子配體結合的能力。其他片段包含N端延伸區及第二包外環則在NMR光譜上沒有明顯的滴定變化。我們進一步將N端片段和ECL2整合到支架蛋白中以評估結合的協同性，發現在滴定實驗中觀察到明顯的光譜訊號變化，推論CXCR3的N端與ECL2具有協同作用來辨識配體CXCL4單體。這項研究可以幫助我們了解CXCL4 / CXCL4L1和CXCR3之間的結合識別位和作用機制。

CXCL4 is a C-X-C-type chemokine, secreted from human platelet. It exhibits a wide range of functions such as hematopoiesis, chemotaxis, and anti-angiogenesis. CXCL4L1 is a nonallelic variant of CXCL4, which differs from CXCL4 in only three residues located in the C-terminal helix. The difference causes a distinct orientation of the C-terminal helix and makes CXCL4L1 more efficient in dissociating from a tetramer into monomers. CXCL4 and CXCL4L1 have been reported to perform their biological functions through activating C-X-C motif chemokine receptor 3 (CXCR3) and the activation is mainly through chemokine monomer formation. The higher tendency of forming monomer is correlated with the higher functional activity in CXCL4L1. In the study, we created two mutants of CXCL4L1-E28K and CXCL4-E28K-V29P-K31P to allow the two chemokines favorably staying in monomeric status. We tested the mutants in binding with different CXCR3 extracellular elements, including N-terminal extension and extra-cellular loop 2 (ECL2). Synthesized peptides were firstly tested to establish the binding preference. The NMR titration experiments only demonstrated sulfated N-terminal extension with chemokine ligand binding ability. The other peptides including the non-sulfated N-terminal extension sequence and ECL2 made no detectable change on NMR spectra. We further integrated the N-terminal extension and ECL2 on a scaffold protein to evaluate the binding cooperativity. The significant perturbation has been observed in the titration experiment. The N-terminal extension together with the ECL2 element synergistically interacts with CXCL4 monomer. The study can help us to understand the recognition selection between CXCL4/CXCL4L1 and CXCR3.

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