Hydrogen Deuterium Exchange Mapping of Chemokine Receptor Complex Interfaces

描述（由申请人提供）：趋化因子是免疫监视、淋巴细胞发育和炎症过程中细胞迁移的关键介质。它们通过与7个跨膜g蛋白偶联受体（gpcr）结合而起作用，引起构象变化，触发细胞内信号通路，参与细胞运动和受体激活。虽然趋化因子被设计为发挥发育和保护作用，但许多疾病是由于这些蛋白的不适当表达、调节或利用而导致的。特异性趋化因子受体为HIV进入细胞提供通道，而其他趋化因子受体则有助于炎症性疾病和许多类型癌症的迁移（转移）。因此，有相当大的兴趣描绘这些蛋白质如何功能的结构细节，以及对抗其功能的机制。目前对配体的结构和受体结合表位有相当多的信息，配体是小的（8-12 kDa）可溶性蛋白。相比之下，对它们的G蛋白偶联受体的结构信息知之甚少。这种信息的缺乏是由于研究膜蛋白的固有困难，特别是真核起源的膜蛋白。表征gpcr的第一个障碍是无法获得足够水平的蛋白质，因为膜蛋白在过度表达时通常是有毒的。用晶体学或核磁共振表征gpcr的结构也明显比可溶性蛋白更难。然而，经过十多年的配体结构-功能研究，我们最近开始关注受体，并有两个受体（D6和CCR1）的水平足以进行生物物理研究。在这一建议中，我们计划开发和应用氢氘交换耦合质谱（DXMS）来表征趋化因子与其受体之间的结合界面。在Aim 1中，我们将优化纯化受体的功能重构。在目标2中，我们将定义趋化因子配体上的受体结合表面。在Aim 3中，我们将开发方法来定义受体上的趋化因子结合表面。除了接触位点外，应该有可能识别受体胞内环的区域，这些区域参与下游信号分子（如G蛋白）的激活，这些分子在稳定性或构象上发生变化。如果成功，该方法将广泛适用于表征趋化因子：受体相互作用。例如，这些研究的结果可用于集中和补充诱变研究。由于CCR1和D6有许多不同的配体，因此最终可以研究不同的配合物；比较激动剂和拮抗剂，特别是关于细胞内环的变化，将是特别有趣的。这些具体目标将通过两个小组的协同活动来实现：Handel博士是趋化因子和受体结构生物学方面的专家，而Woods博士是DXMS方法方面的专家，他对开发膜蛋白的DXMS方法特别感兴趣，并且已经参与其中。公共卫生相关性：在正常生理条件下，趋化因子及其受体在免疫系统功能和发育的背景下参与以控制细胞迁移能力为中心的过程。然而，不适当的趋化因子介导的细胞迁移和炎症导致或促成许多疾病的病理，如哮喘、类风湿关节炎、多发性硬化症、心脏病和癌症。发展像DXMS这样的方法来了解趋化因子：受体相互作用和功能的分子细节，可能有助于设计用于许多疾病的小分子和蛋白质疗法。

DESCRIPTION (provided by applicant): Chemokines are critical mediators of cell migration during immune surveillance, lymphocyte development, and inflammation. They function by binding to seven transmembrane G-protein coupled receptors (GPCRs), causing conformational changes that trigger intracellular signaling pathways involved in cell movement and receptor activation. Although chemokines were designed to carry out developmental and protective roles, many diseases result from inappropriate expression, regulation, or utilization of these proteins. Specific chemokine receptors provide the portals for HIV to get into cells, while others contribute to inflammatory disease, and migration (metastasis) of many types of cancers. Thus, there is considerable interest in delineating the structural details of how these proteins function, and mechanisms for antagonizing their function. Presently there is a fair amount of information on the structure and receptor binding epitopes of the ligands, which are small (8-12 kDa) soluble proteins. By contrast, very little structural information is known about their G protein-coupled receptors. This lack of information is due to the inherent difficulties in studying membrane proteins, particularly those of eukaryotic origin. The first obstacle in characterizing GPCRs has been the inability to obtain sufficient levels of protein because membrane proteins are usually toxic when over- expressed. Structural characterization of GPCRs by crystallography or NMR is also significantly more difficult than for soluble proteins. However, after more than a decade of structure-function studies of ligands, we recently began focusing on the receptors and have two receptors (D6 and CCR1) at levels sufficient for biophysical studies. In this proposal, we plan to develop and apply Hydrogen Deuterium Exchange coupled with Mass spectrometry (DXMS) to characterize the binding interfaces between chemokines and their receptors. In Aim 1, we will optimize the functional reconstitution of purified receptor. In Aim 2, we will define the receptor-binding surface on the chemokine ligand(s). In Aim 3, we will develop methods to define the chemokine-binding surface on the receptors. In addition to contact sites, it should be possible to identify regions of the intracellular loops of the receptor that are involved in activation of downstream signaling molecules like G proteins, which undergo changes in stability or conformation. If successful, the methods should have broad applicability for characterizing chemokine:receptor interactions. For example, the results of these studies can be used to focus and complement mutagenesis studies. Since there are many different ligands of CCR1 and D6, different complexes can ultimately be investigated; comparison of agonists and antagonists, especially with respect to changes in intracellular loops, will be particularly interesting. These specific aims will be accomplished by the synergistic activities of two groups: Dr. Handel, an expert in the structural biology of chemokines and receptors, and Dr. Woods, an expert in DXMS methodology, who is particularly interested, and already involved, in developing DXMS methods for membrane proteins. PUBLIC HEALTH RELEVANCE: Under normal physiological conditions, chemokines and their receptors are involved in processes centered around their ability to control cell migration in the context of immune system function and development. However, inappropriate chemokine-mediated cell migration and inflammation causes or contributes to the pathology of many diseases such as asthma, rheumatoid arthritis, multiple sclerosis, heart disease, and cancer. Developing methods like DXMS to understand the molecular details of chemokine:receptor interactions and function, may facilitate the design of small molecule and protein therapeutics for many diseases.