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类淋巴细胞募集的结构和生化基础，这是一种通过在两个完全不同的三级结构之间相互转换而打破传统范式的趋化因子。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地激活了一组20种G蛋白偶联受体的成员。这些分泌的信号蛋白也结合细胞外基质糖胺聚糖（GAG），以指导沿趋化因子浓度梯度的迁移。淋巴趋化因子（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选择性募集T和NK细胞。在肿瘤消退和组织移植排斥中的功能作用突出了两种疾病状态，可能对Ltn模拟物或拮抗剂治疗有反应。Ltn仅包含所有其他趋化因子中保守的两个二硫化物中的一个，并且进一步区分为独特的无序c端延伸，这是活性所必需的。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的实验将使用核磁共振波谱和诱变来确定Ltn的新型非趋化因子结构，测量相互转换的动力学，并验证其构象平衡源于缺乏保守的二硫桥的假设。Aim 2的目标是通过表面等离子体共振改变构象平衡并测量每个物种的结合亲和力，以验证结构相互转换产生高亲和力的GAG结合位点对体内活性至关重要的假设。在Specific Aim 3中，我们将探讨基本c端的保守残基在GPCR激活中的作用，并比较体外和体内构象受限Ltn变异的活性，以验证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的目的是为在体内调节淋巴细胞运输由淋巴蛋白指导开辟新的途径。

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.