From the Department of Microbiology & Immunology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, United States.
Department of Biomedical Sciences, University of Copenhagen, Copenhagen 2200, Denmark.
Biochemistry. 2020 Apr 7;59(13):1338-1350. doi: 10.1021/acs.biochem.0c00047. Epub 2020 Mar 26.
The chemokines CCL21 and CCL19, through binding of their cognate receptor CCR7, orchestrate lymph node homing of dendritic cells and naïve T cells. CCL21 differs from CCL19 via an unstructured 32 residue C-terminal domain. Previously described roles for the CCL21 C-terminus include GAG-binding, spatial localization to lymphatic vessels, and autoinhibitory modulation of CCR7-mediated chemotaxis. While truncation of the C-terminal tail induced chemical shift changes in the folded chemokine domain, the structural basis for its influence on CCL21 function remains largely unexplored. CCL21 concentration-dependent NMR chemical shifts revealed weak, nonphysiological self-association that mimics the truncation of the C-terminal tail. We generated a series of C-terminal truncation variants to dissect the C-terminus influence on CCL21 structure and receptor activation. Using NMR spectroscopy, we found that CCL21 residues 80-90 mediate contacts with the chemokine domain. In cell-based assays for CCR7 and ACKR4 activation, we also found that residues 92-100 reduced CCL21 potency in calcium flux, cAMP inhibition, and β-arrestin recruitment. Taken together, these structure-function studies support a model wherein intramolecular interactions with specific residues of the flexible C-terminus stabilize a less active monomer conformation of the CCL21. We speculate that the autoinhibitory intramolecular contacts between the C-terminal tail and chemokine body are disrupted by GAG binding and/or interactions with the CCR7 receptor to ensure optimal functionality.
趋化因子 CCL21 和 CCL19 通过与其同源受体 CCR7 的结合，协调树突状细胞和幼稚 T 细胞归巢到淋巴结。CCL21 与 CCL19 的区别在于其无结构的 32 个残基 C 末端结构域。先前描述的 CCL21 C 末端的作用包括 GAG 结合、空间定位到淋巴管以及对 CCR7 介导的趋化作用的自动抑制调节。虽然 C 末端尾巴的截断诱导了折叠趋化因子结构域的化学位移变化，但它对 CCL21 功能的影响的结构基础在很大程度上仍未得到探索。CCL21 浓度依赖性 NMR 化学位移揭示了弱的、非生理的自缔合，模拟了 C 末端尾巴的截断。我们生成了一系列 C 末端截断变体，以剖析 C 末端对 CCL21 结构和受体激活的影响。使用 NMR 光谱学，我们发现 CCL21 残基 80-90 介导与趋化因子结构域的接触。在用于 CCR7 和 ACKR4 激活的基于细胞的测定中，我们还发现残基 92-100 降低了 CCL21 在钙通量、cAMP 抑制和β-抑制蛋白募集中的效力。总之，这些结构功能研究支持这样一种模型，即与柔性 C 末端的特定残基的分子内相互作用稳定 CCL21 的较少活性单体构象。我们推测，C 末端尾巴和趋化因子体之间的自动抑制分子内接触被 GAG 结合和/或与 CCR7 受体的相互作用破坏，以确保最佳功能。
