Conformational duality in the human chemokine Ltn/XCL1

描述（由申请人提供）：这项工作的目标是了解人淋巴细胞趋化素募集淋巴细胞的结构和生化基础，人淋巴细胞趋化素是一种趋化因子，通过在两种完全不同的三级结构之间相互转换来挑战传统范例。脊椎动物免疫系统中的炎症是由大约 50 种趋化因子精心策划的，这些趋化因子特异性激活一组 20 G 蛋白偶联受体的成员。这些分泌的信号蛋白还结合细胞外基质糖胺聚糖（GAG），以引导沿着趋化因子浓度梯度迁移。淋巴趋化蛋白 (Ltn/XCL1) 是 C 类趋化因子的原型和单一成员，通过其同源受体 XCR1 选择性地募集 T 和 NK 细胞。肿瘤消退和组织移植排斥中的功能作用凸显了两种可能对 Ltn 模拟物或拮抗剂治疗有反应的疾病状态。 Ltn 仅包含所有其他趋化因子中保守的两个二硫键之一，并通过对活性至关重要的独特的无序 C 末端延伸进一步区分。这些不同序列特征的结果是，Ltn 在生理溶液条件下同时采用两种不同的三级结构，其中只有一种类似于典型的趋化因子折叠。具体目标 1 中的实验将使用 NMR 光谱和诱变来确定 Ltn 的新型非趋化因子结构，测量相互转化的动力学，并测试其构象平衡源自缺乏保守二硫键的假设。目标 2 的目标是通过表面等离子共振改变构象平衡并测量每个物种的结合亲和力，以测试结构互变产生体内活性必需的高亲和力 GAG 结合位点的假设。在具体目标 3 中，将探究必需 C 末端的保守残基在 GPCR 激活中的作用，并比较构象限制性 Ltn 变体的体外和体内活性，以检验只有趋化因子样结构才能结合和激活受体的假设。这些结构和生化研究旨在为淋巴因子引导的体内淋巴细胞运输调节开辟新途径。

DESCRIPTION (provided by applicant): The goal of this work is to understand the structural and biochemical basis for the recruitment of lymphocytes by human Lymphotactin, a chemokine that defies the traditional paradigm by interconverting between two entirely different tertiary structures. Inflammation in the vertebrate immune system is orchestrated by approximately 50 chemokines that specifically activate members of a group of 20 G protein-coupled receptors. These secreted signaling proteins also bind extracellular matrix glycosaminoglycans (GAG) in order to direct migration along a gradient of chemokine concentration. Lymphotactin (Ltn/XCL1), the prototype and single member of the C class of chemokines, acts through its cognate receptor XCR1 to selectively recruit T and NK cells. Functional roles in tumor regression and tissue transplant rejection highlight two disease states that may respond to treatment with either Ltn mimetics or antagonists. Ltn contains only one of the two disulfides conserved in all other chemokines, and is further distinguished by a unique disordered C-terminal extension that is essential for activity. A consequence of these divergent sequence features is that Ltn simultaneously adopts two distinct tertiary structures in physiological solution conditions, only one of which resembles the canonical chemokine fold. Experiments in Specific Aim 1 will use NMR spectroscopy and mutagenesis to determine the novel non-chemokine structure of Ltn, measure the dynamics of interconversion, and test the hypothesis that its conformational equilibrium derives from the lack of a conserved disulfide bridge. The goal of Aim 2 is to alter the conformational equilibrium and measure binding affinities for each species by surface plasmon resonance, in order to test the hypothesis that the structural interconversion creates a high affinity GAG binding site essential for in vivo activity. In Specific Aim 3, conserved residues in the essential C-terminus will be probed for their role in GPCR activation, and comparisons of activity in vitro and in vivo for conformationally-restricted Ltn variants will be compared in order to test the hypothesis that only the chemokine-like structure is competent to bind and activate the receptor. These structural and biochemical studies are designed to open new avenues for in vivo modulation of lymphocyte trafficking directed by Lymphotactin.