Conformational duality in the human chemokine Ltn/XCL1

描述(申请人提供)：这项工作的目标是了解人类淋巴肌动蛋白招募淋巴细胞的结构和生化基础，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挑战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种趋化因子协调的，这些趋化因子专门激活一组20G蛋白偶联受体的成员。这些分泌的信号蛋白还与细胞外基质糖胺多聚糖(GAG)结合，以引导趋化因子浓度梯度的迁移。淋巴细胞趋化蛋白(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选择性地招募T细胞和NK细胞。在肿瘤消退和组织移植排斥反应中的功能作用突出了两种疾病状态，它们可能对LTN模拟物或拮抗剂的治疗有反应。LTN只包含在所有其他趋化因子中保守的两个二硫键中的一个，并进一步通过独特的无序C末端延伸而区分开来，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个不同的三级结构，其中只有一个类似于典型的趋化因子折叠。在特定目标1的实验中，将使用核磁共振光谱和诱变来确定LTN的新的非趋化因子结构，测量相互转化的动力学，并验证其构象平衡来自于缺乏保守的二硫键的假设。目标2的目标是改变构象平衡，并通过表面等离子体共振测量每个物种的结合亲和力，以验证结构相互转换产生对体内活性至关重要的高亲和力Gag结合位点的假设。在特定目标3中，将探讨基本C末端的保守残基在GPCR激活中的作用，并将比较构象受限的LTN变体的体外和体内活性，以验证只有趋化素样结构才有能力结合和激活受体的假设。这些结构和生化研究旨在为淋巴肌动蛋白在体内调节淋巴细胞转运开辟新的途径。

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.